Evaluation of miniaturized Illumina DNA preparation protocols for SARS-CoV-2 whole genome sequencing protocol

Prepare the cDNA master mix in the pre-PCR clean room. The master mix hood must be decontaminated before and after use with 10% extran and 70% ethanol, and sterilised with ultraviolet light (UV).

Mix the following components in a labeled 1.5ml tube:

A	B
Component	Volume (ul)
LunaScript	1
Template RNA	5.5
Total	6.5

Table 1. cDNA synthesis master-mix 1

Add 1 µl of LunaScript reagent into each well of a 96-well nested plate.

Transfer 5,5 ul of total nucleic acid extracts to each well as per Table 1.

The total volume in the tube should now be 6.5 µl.

Incubate the reaction as follows in a thermal cycler.

A	B
Temperature (°C)	Time
25 °C	2 minutes
55 °C	10 minutes
95 °C	1 minute
4	Hold

Table 2. PCR conditions – cDNA synthesis

Primers must be diluted and pooled in a clean master mix hood using nuclease-free water. The master mix hood must be decontaminated with 10% extran and 70% ethanol before and after use, and sterilised with ultraviolet light (UV).

                                                                                                                                                                                                                                                                                                                                                                                                                         To generate overlapping 400 nucleotide amplicons for this protocol, 2019-nCoV primers were designed using the Primal Scheme.

Resuspend lyophilised primers at a concentration of 100 M each if necessary.

To generate 100 µM primer pool stocks , add 5 µl of each primer pair (named pool 1 or pool 2) to a 1.5ml eppendorf tube labeled either “ Pool 1 (100μM) ” or “ Pool 2 (100μM) ”. Total volume will be 490 µl for Pool 1 (100uM) and 490 µl for Pool 2 (100uM). These are now 100 µM stocks of each primer.

To make 10M primer stocks, dilute the 100M primer pool 1:10 in molecular-grade water.

It is advised to make multiple aliquots of each primer pool in case of degradation or contamination.

Prepare the PCR mastermix in the clean mastermix room.

The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).

A master mix for each pool must be made up in the master mix hood.

Mix the following components in a labelled 1.5ml eppendorf tube:

A	B	C
Component	Pool 1 volumes (μl)	Pool 2 volumes (μl)
5X Q5 Reaction Buffer	2.5	2.5
10mM dNTPs	0.25	0.25
Q5 Hot Start DNA Polymerase	0.125	0.125
Primer Pool 1 or 2 (10μM)	1.8	1.8
Nuclease-free water	5.4	5.4
Total	10	10

Table 3. PCR COVID-19 tiling PCR mastermix

Make a master mix by multiplying each component by the number of samples being tested.

Add 2.5 µl 5X Q5 Reaction Buffer to a labeled 1.5ml eppendorf tube.

Add 0.25 µl 10mM dNTPs.

Add 0.125 µl Q5 Hot Start DNA Polymerase.

Add 1.8 µl Primer Pool 1 or 2 (10μM).

Add 5.4 µl Nuclease-free water.

Aliquot 10 µl mastermix in labelled PCR strip tubes.

Add 2.5 µl of cDNA under the extraction hood or general lab hood, which has been decontaminated using 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).

Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.

Incubate the reaction as follows in a thermal cycler.

A	B	C	D
Step	Temperature (°C)	Time	Cycles
Heat Activation	98	30 seconds	1
Denaturation	98	15 seconds	35
Annealing	65	5 minutes
Hold	4	∞

Table 4. Tiling PCR conditions *Cycle number should be 25 for Ct 18-21, and up to a maximum of 35 cycles for Ct 35.

Combine the entire contents of “ Pool 1 ” and “ Pool 2 ” PCR reactions for each biological sample into a single tube.

Add 5 µl undiluted DNA to each well of a 96-well PCR plate / 0.2ml strip tubes.

A	B	C
BLT(bead-linked transposomes)	2°C to 8°C	Bring to room temperature. Vortex to mix. Do not centrifuge before pipetting.
TB1(Tagmentation buffer)	-25°C to -15°C	Bring to room temperature. Vortex to mix.

Table 5. Preparation of reagents

Vortex BLT vigorously for 00:00:10 to resuspend.

Vortex in between adding BLT as necessary.

Prepare the tagmentation master mix.

A	B
Component	Volume (μl) per sample
BLT	1
TB1	1
Total	2

Table 6. Tagmentation Master Mix

Please scale this step as needed.

For 1 sample:

1 µl BLT

1 µl TB1

---

2 µl Total

Vortex the tagmentation master mix thoroughly.

Transfer 2 µl tagmentation master mix to each well of the plate containing sample.

Use fresh tips for each sample column.

Resuspend by pipetting each sample 10 times.

Seal the plate with a plate sealer, place on the preprogrammed thermal cycler, and run the tagmentation program.

A	B
Temperature (°C)	Time
55	15 minutes
10	Hold

Table 7. PCR – Tagmentation conditions

A	B	C
Item	Storage	Instructions
TSB (Tagment stop buffer)	15 oC to 30 oC	If precipitates are observed, heat at 37°C for 10 minutes, and then vortex until precipitates are dissolved. Use at room temperature.

Table 8. Preparation of Reagents

Add 1 µl TSB to the tagmentation reaction.

Resuspend the beads by slowly pipetting each well/ tube 10 times.

Seal the plate with / tubes, place it on the preprogrammed thermal cycler, and run the stop tag programme.

A	B
Temperature (°C)	Time
37	15 minutes
10	Hold

Table 9. Stop-Tag programme

Place the plate on the magnetic stand for approximately 00:01:00 until liquid is clear.

Using a multichannel pipette, remove and discard supernatant.

Remove the sample plate from the magnetic stand and add 10 µl TWB directly onto the beads using a deliberately slow pipetting technique.

To avoid incorrect volume aspiration and incomplete mixing, this slow pipetting technique reduces the possibility of TWB foaming.

Slowly pipette until beads are fully resuspended.

Place the plate on the magnetic stand for approximately 00:01:00 until liquid is clear.

Using a multichannel pipette, remove and discard supernatant.

Remove the plate from the magnetic stand and add 10 µl TWB directly onto the beads using a deliberately slow pipetting technique.

Slowly pipette each well/tube to resuspend the beads.

Place the plate on the magnetic stand for approximately 00:01:00 until liquid is clear.

Using a multichannel pipette, remove and discard supernatant.

Remove the plate from the magnetic stand and use a deliberately slow pipetting technique to add

10 µl TWB directly onto the beads.

Slowly pipette each well/tube to resuspend the beads.

Seal the plate and keep on the magnetic stand until step 47 of the Procedure section in Amplify Tagmented DNA.

The TWB remains in the wells to prevent overdrying of the beads.

A	B	C
Item	Storage	Instructions
EPM (Enhanced PCR Mix)	-25°C to-15°C	Thaw on ice. Invert to mix, then briefly centrifuge.
Index Adapters Tubes/plates	-25°C to-15°C	Thaw at room temperature. [Tubes] Vortex to mix, then centrifuge briefly. [Plates] Spin briefly before use.

Table 10. Preparation of Reagents

A	B
Component	Volume (μl) per sample
EPM	2
NFW (Nuclease Free Water)	2
Total	4

Table 11. PCR Master Mix The following volumes were combined to prepare the PCR master mix (Table 13). Each volume was multiplied by the number of samples being processed. Reagent overage was included in the volume to ensure accurate pipetting.

Vortex and centrifuge the PCR master mix at 280 x g 00:00:10.

With the plate on the magnetic stand, use a multichannel pipette to remove and discard the supernatant. (from step 42 of post-tagmentation clean-up).The foam that remains on the walls of the well does not adversely affect the library.

Remove from the magnet.

Immediately add 4 µl PCR master mix directly onto the beads in each sample well/ tube.

Pipette mix until the beads are fully resuspended. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 00:01:00.

Seal the sample plate and centrifuge at 280 x g 00:00:30.

Add 1 µl of the appropriate index adapters to each sample.

Using a pipette set to 4 μl, pipette 10 times to mix. Alternatively, seal the plate/ tubes and use a plate shaker at 1600 rpm for 00:01:00.

Centrifuge at 280 x g 00:00:30.

Place on the thermal cycler and run the Enrichment PCR program.

A	B	C
Temperature (°C)	Time
68°C	3 minutes
98°C	3 minutes
98°C	45 seconds	8 cycles
62°C	30 seconds
68°C	2 minutes
68°C	1 minute
10°C	Hold

Table 12. Enrichment PCR Conditions

SAFE STOPPING POINT

If you are stopping, store at 2 °C to 8 °C for up to 72:00:00 (3 days).

A	B	C
Item	Storage	Instructions
SPB (Sample Purification Beads)	2°C to 8°C	Let stand at room temperature for 30 minutes. Vortex and invert to mix.
RSB (Resuspension Buffer)	-25°C to-15°C	Thaw and bring to room temperature. Vortex to mix.

Table 13. Preparation of Reagents

Prepare fresh 80 % EtOH from absolute ethanol.

Centrifuge at 280 x g 00:01:00 to bring all contents to the bottom.

Place the plate/ tubes on a magnetic stand for approximately 00:01:00 until liquid is clear.

Transfer 4.5 µl supernatant from each well of the PCR plate/ tubes to the corresponding well of a new plate/ tubes.

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Add 4 µl nuclease-free water to each well/ tube.                                                                                                                                                                                               

Vortex and invert SPB multiple times to resuspend

Add 4.5 µl SPB to each well/ tube.

Mix well by pipetting 10 times. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 00:01:00.

Incubate at Room temperature for 00:01:00.

Place on the magnetic stand for approximately 00:01:00 until the liquid is clear. During incubation, thoroughly vortex the SPB (undiluted stock tube), and then add 1.5 µl to each well of a new plate/tubes.

Transfer 12.5 µl supernatant from each well of the first plate/ tubes into the corresponding well of the second plate/ tubes (containing 1.5 μl undiluted SPB).

Mix well by pipetting 10 times. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 00:01:00.

Discard the first plate/ tubes.

Pipette each well 10 times to mix.

Incubate at Room temperature for 00:01:00.

Place on the magnetic stand for approximately 00:01:00 until the liquid is clear.

Without disturbing the beads, remove and discard supernatant.

Wash two times as follows:

Add 20 µl freshly prepared 80% ethanol with the plate on the magnetic stand.

Incubate for 00:00:30.

Without disturbing the beads, remove and discard the supernatant.

Add 20 µl freshly prepared 80% ethanol with the plate on the magnetic stand.

Without disturbing the beads, remove and discard the supernatant.

Use a 20 μl pipette to remove any residual ethanol.

Air-dry on the magnetic stand for 00:01:00.

Remove from the magnetic stand.

Add 13.5 µl RSB to each well/ tube.

Resuspend by pipette mixing.

Incubate at Room temperature for 00:01:00.

Place the plate/ tubes on the magnetic stand for approximately 00:01:00.

Transfer 12 µl supernatant to a new 96-well PCR plate/ tubes.

Rapid Method:

Using an example of 96 samples:

Pool 4.5ul of each sample after the enrichment step into a 1.5 µl tube= 432 µl (4.5 x 96).

Add 384 µl (4x 96) of nuclease free water.

Add 432 µl (4.5 X96) of SPB.

Vortex and incubate for 5 minutes.

Place on amagnetic rack for 3 minutes.

Transfer the supernatant to a clean 1.5 µl tube.

Add 144 µl (1.5 x 96) of SPB.

Vortex and incubate for 5 minutes.

Place on a magnetic rack for 3 minutes.

Wash twice with 1 ml of 80% ethanol.

Remove all traces of ethanol after the last wash.

Elute in 35 µl of resuspension buffer.

SAFE STOPPING POINT

If you are stopping, seal the plate, and store at -25 °C to -15 °C for up to 30 days.

Using the Qubit concentrations and fragment length normalize the libraries to equimolar 4nM by diluting with RSB buffer.

Calculate theappropriate amount of diluent on an excel sheet to add to respective sample libraries in order to achieve a 4nm library concentration, using the following formula:

Nanomolar concentration = (ng/μl /660 x 500) x 10^6

Pipette mix 5 times.

Use a multi-channel pipette to transfer 5 µl of the diluted sample library to an 8 strip-tube and spin briefly.

Pool the library samples from the 8-strip tubes to a labelled Pooled Amplicon Library (PAL) 2ml eppendorf tube.

Proceed to library denaturation.

Remove the tube of HT1 (Hybridization Buffer) from the freezer (-15°C to -25°C) and set aside at Room temperature to thaw.

When thawed, store at 2 °C to 8 °C until you are ready to dilute denatured libraries.µL

Prepare 500μl of 0.2 N NaOH by combining the following volumes in a 1.5ml microcentrifuge tube: 490 µl laboratory-grade water and 10 µl Stock 1.0 N NaOH.

Refer to the formula below: 1M=1N1M=1N 10N(x)=(0.2)(500)10N(x)=(0.2)(500) x = 10 µl NaOH + 490 µl laboratory-grade water.

A fresh dilution of 0.2N NaOH is required for the denaturation process in preparing sample DNA and a PhiX control.

Invert the tube several times to mix.

Combine the following volumes of pooled sample DNA and freshly diluted 0.2 N NaOH in a micro-centrifuge tube, by adding 5 µl of 4nM sample DNA to 5 µl of 4nM sample DNA.

Discard the remaining dilution of 0.2 N NaOH or set aside to prepare a PhiX control within the next 12:00:00.

Vortex briefly to mix the sample solution, and then centrifuge the sample solution to 280 x g 00:01:00.

Incubate for 00:05:00 at Room temperature to denature the DNA into single strands.

Add 990 µl of pre-chilled HT1 to the 10 µl of 4nM denatured library. The result is a 20pM denatured library in 1 mM NaOH.

Place the denatured DNA On ice or at 4 °C until you are ready to proceed to the final dilution.

Use the following instructions to dilute the 20pM DNA further to give 600μl of the desired input concentration.

Dilute the denatured DNA to the desired concentration using the following example (if using 1% PhiX):

A	B	C	D
Final Concentration	20pM denatured DNA	1% PhiX	Pre-chilledHT1
12 pM	354.6μl	3.6μl	240μl

Table 14. Dilution of denatured library to desired concentration *This was found to be the optimal loading concentration when using a Miseq V2 500 cycle kit

Invert several times to mix and then pulse centrifuge.

To dilute PhiX to 4nM concentration, combine the following volumes in a microcentrifuge tube: • 2 µl of 10nM PhiX library • 3 µl of 10mM Tris-Cl, pH8.5 with 0.1 % Tween 20 If not prepared within the last 12 hours, prepare a fresh dilution of 0.2 N NaOH.

Combine the following volumes in a micro-centrifuge tube: • 5 µl of 4 nM PhiX library • 5 µl of 0.2 N NaOH

Vortex briefly to mix.

Centrifuge at 280 x g 00:01:00.

Incubate at Room temperature for 00:05:00.

Dilute denatured PhiX to 20pM by adding pre-chilled HT1 to the denatured PhiX library as follows:

990 µl pre-chilled HT1 to 10 µl denatured PhiX library •

Invert to mix.

Combine Library and PhiX Control.

Mix this solution well and briefly centrifuge.

Keep On ice or at 4 °C until it is ready to be loaded onto the MiSeq reagent cartridge.