Fast-S: Single tube amplification and PCR barcoding of SARS-CoV-2 S gene for Nanopore sequencing

Place PCR tubes with caps in a 96 sample rack for pool A and pool B amplification step.

Label a set of PCR tube caps for amplification using the pool A and pool B according to the Oxford Nanopore PCR Barcoding Expansion 1-96 (stock 10 μM).

A	B	C	D	E	F	G	H	I	J	K	L	M
A	BC01	BC02	BC03	BC04	BC05	BC06	BC07	BC08	BC09	BC10	BC11	BC12
B	BC13	BC14	BC15	BC16	BC17	BC18	BC19	BC20	BC21	BC22	BC23	BC24
C	BC25	BC26	BC27	BC28	BC29	BC30	BC31	BC32	BC33	BC34	BC35	BC36
D	BC37	BC38	BC39	BC40	BC41	BC42	BC43	BC44	BC45	BC46	BC47	BC48
E	BC49	BC50	BC51	BC52	BC53	BC54	BC55	BC56	BC57	BC58	BC59	BC60
F	BC61	BC62	BC63	BC64	BC65	BC66	BC67	BC68	BC69	BC70	BC71	BC72
G	BC73	BC74	BC75	BC76	BC77	BC78	BC79	BC80	BC81	BC82	BC83	BC84
H	BC85	BC86	BC87	BC88	BC89	BC90	BC91	BC92	BC93	BC94	BC95	BC96

Table 1: Oxford Nanopore PCR Barcoding Expansion 1-96 tube labeling for pool A and pool B amplification step

Spin down briefly the PCR Barcoding Expansion 1-96 tubes and open each carefully avoiding spray generation. Transfer 1 μL of each barcode using a multichannel pipette to the PCR tube cap and incubate 1hr at 37 ºC or at room temperature until the drop has dried.

Store in a dry place until use.

Keeping the SARS-CoV-2 extracted RNA samples on ice all the time and spin down the tubes.

Set up the RT-PCR reaction tubes in a clean pre-PCR cabinet by adding 2 μL of LunaScript® RT SuperMix to each PCR tube. Include a RT-PCR negative control by replacing RNA sample with nuclease free water.

Add 8 uL of sample to the tube containing the LunaScript® RT SuperMix and mix gently. The final volume of the reaction is 10 μL.

Incubate in a thermal cycler using the the following instructions:

A	B	C	D
Primer annealing	25	2 min	1
cDNA Synthesis	55	10 min
Heat inactivation	95	1 min
Hold	10	-

Table 2: SARS-CoV-2 RT-PCR thermal profile.

Set up the first round PCR reaction in a pre-PCR cabinet for primer pool A and primer pool B

A	B	C
Nuclease free water	400 μL	400 μL
Primer pool A 30 μM)	25 μL	--
Primer pool B (30 μM)	--	25 μL
LongAmp® Taq 2X Master Mix	625 μL	625 μL
Final volume	1050 μL	1050 μL

Table 3: S gene PCR multiplex amplification master mixes for pool A and B.

Transfer 10,5 μL of the PCR Master mix Pool A to the PCR tube set A and 10,5 μLof the PCR Master mix Pool B to the PCR tube set B, respectively.

Using a multichannel pipette, transfer 2 μL of reversed transcribed product from the "Reverse Transcription" section to the corresponding Pool A and Pool B PCR strip tubes. Carry over the negative and positive controls.

Spin down briefly and replace the PCR strip caps with the PCR tube caps containing the immobilized Barcoding Expansion 1-96 barcodes as described in the "Barcode immobilization" section.

Incubate in the thermal cycler with the following program:

A	B	C	D
Initial denaturation	94	30 s	1
Denaturation	94	15 s	20
Annealing and extension	63	3 m
Hold	10	--	--

Table 4: S gene PCR amplification using the LongAmp™ Taq2X polymerase.

Remove the PCR strips off the thermal cycler and invert the tubes at least 10 times until the immobilized PCR barcodes in the PCR strip caps are dissolved in the PCR master mix from the amplification step. Spin down and return the tubes into the thermal cycler and proceed to the barcoding step:

A	B	C	D
Initial denaturation	95	3 m	1
Denaturation	95	15 s	15
Annealing	62	15 s
Extension	65	50 s
Final extension	65	10 m	1
Hold	10	--	--

Table 5: S gene PCR barcoding of S gene amplified material using the amplification PCR master mix from the amplification step

Spin down the tubes and pool all samples in a 1.5 mL LoBind tube.

Add 0.5X volume of Ampure XP beads. Incubate 5 minutes in a rotator mixer. Spin down and rest the tubes in a magnetic rack for 5 minutes.

Discard the supernatant by aspiration, taking care not to disturb the pellet beads.

Wash the beads with Ethanol 70%. Repeat this step.

Let the pellet air dry for ~30 seconds and add 50 uL of nuclease free water. Incubate for 2 minutes at room temperature, spin down and place the PCR tubes in the magnetic rack for 5 minutes.

Recover the supernatant.

Prepare 1 µg of clean barcoded pool in 48 µL.

Prepare the end prep mix as follows:

A	B
NEBNext FFPE DNA Repair Mix	2
Ultra II End-prep enzyme mix	3
NEBNext FFPE DNA Repair Buffer	3.5
Ultra II End-prep reaction buffer	3.5
Clean barcoded pool (1 µg)	48
Final volume	60

Table 6: End prep reaction mix.

Incubate the End-prep mix in the thermal cycler as follows:

A	B	C
Enzymes incubation	20	5 min
Enzymes inactivation	65	5 min

Table 7: End-prep thermal incubation

[Optional] Add 1X volume of AMPure XP beads to the reaction and incubate in the rotator mixer for 5 minutes. Spin down and rest the tubes in the magnetic rack for 5 minutes. Discard the supernatant by aspiration, taking care not to disturb the pellet beads. Let the pellet air dry for ~30 seconds and add 61 µL of nuclease free water and incubate at room temperature for 2 minutes.Spin down and rest the tubes in the magnetic rack for 5 minutes and recover the supernatant. Spin down and rest the tubes in the magnetic rack for 5 minutes and recover the supernatant.

Prepare the following adapter ligation mix:

A	B
Clean end-prepped DNA	60
Ligation Buffer (LNB)	25
NEBNext Quick T4 DNA Ligase	10
AMX adapter	5
Final volume	100

Table 8: Adapter (AMX) ligation mix.

Incubate the reaction for 10 minutes at room temperature.

Spin down the tubes and add AMPure XP beads 0.4X volume. Incubate in the rotator mixer for 5 minutes at room temperature.

Spin down the tubes and rest the tubes in the magnet rack for 5 minutes. Pipette off the supernatant.

Add 200 µL of Short Fragment Buffer (SFB) and mix gently. Spin down and place the tube in the magnet rack for 5 minutes. Discard the supernatant.

Repeat the previous step.

Remove the tubes from the magnet and add 15 µL of Elution Buffer (EB). Flick the tube, spin down briefly and incubate the tube for 10 minutes at 37 ºC.

Quantify the final library using a fluorometric assay.

Use the Nanopore standard procedure for priming the FLO-MIN106D or FLO-FLG001 flow cells.

https://community.nanoporetech.com/nanopore_learning/lessons/priming-and-loading-your-flow-cell

Once the flow cell is correctly primer proceed to load the library mixing the following:

A	B
Sequencing Buffer (SQB)	37.5
Loading Beads (LB)	25.5
DNA library (~300 ng)	12
Final volume	75

Table 9: DNA library to load in a FLO-MIN106D.

A	B
Sequencing Buffer (SQB)	15
Loading Beads (LB)	10
DNA library (~200 ng)	5
Final Volume	30

Table 9: DNA library to load in a FLO-FLG001.

Use epi2me-labs/wf-artic V1 scheme for consensus generation

Software

Value	Label
wf-artic	NAME
https://github.com/epi2me-labs/wf-artic	LINK

hedgehog for lineage set assignment using maximum ambiguity

Software

Value	Label
hedgehog	NAME
https://github.com/cov-lineages/hedgehog	LINK

Use president for S gene completeness

Software

Value	Label
president	NAME
https://gitlab.com/RKIBioinformaticsPipelines/president	LINK

and samtools for average sequencing depth

Software

Value	Label
samtools	NAME
https://github.com/samtools/samtools	LINK

Run the sequencing experiment until reaching at least 300X of average sequencing depth and or more than 90% of S gene completeness for optimal results.