SARS-CoV-2 Whole Genome Sequencing on Illumina

Guerrino Macori, Lauren Russell, Seamus Fanning

Published: 2023-07-20 DOI: 10.17504/protocols.io.brfzm3p6

Disclaimer

In development

We are still developing and optimizing this protocol. Comments and feedback appreciated.

Abstract

This SOP describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently obtaining, through the amplicons tiling, the whole viral genome using V3 nCov-2019 primers (ARTIC). This is followed by library construction and pooling of samples and quantitation, prior to sequencing on the Illumina MiSeq.

The SOP is adapted from the nCoV-2019 sequencing protocol: https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w, and it was used in this study:

Lucey M, Macori G, Mullane N, Sutton-Fitzpatrick U, Gonzalez G, Coughlan S, Purcell A, Fenelon L, Fanning S, Schaffer K. Whole-genome Sequencing to Track Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmission in Nosocomial Outbreaks. Clinical Infectious Diseases. 2020.

https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1433/5909421

Steps

SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription

cDNA/Reverse Transcription Section Date/Initials:_________________

In this section, the nucleic acid is extracted and used for the qPCR diagnostic test as starting material for sequencing.

1.1.

[ ] In a PCR hood, mix the following reagents in a 0.2mL PCR tube or PCR plate:

A	B	C
Reagent	Volume (µL)	MM for N+2 samples
60 µM random hexamers	1.0
10 mM dNTPs mix (10 mM each)	1.0
Template RNA	11.0
Total	13.0

Master mix calculations

Note

Mastermix should be made up in the mastermix cabinet and aliquoted into PCR tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.Each reaction should have 13µL when mixed. If using master mix, it is recommended to add the of the master mix to the PCR tube/plate first, then add the 11 µl of RNA to help prevent contamination.

Lot# _______________ Exp. Date _______________

1.2.

[ ] Mix gently and briefly centrifuge to spin down the components, and return On ice.

1.3.

[ ] Preheat Thermocycler to 65°C, with heated lid at 105°C

1.4.

[ ] Incubate the reaction at 65°C for 0h 5m 0s, followed by an immediate snap-cool On ice for at least 0h 1m 0s.

1.5.

[ ] In a clean 1.5mL LoBind tube (96 well plates can also be used), mix together the following reagents:

A	B	C
Reagent	Volume (uL)	MM for N+2 samples
SuperScript IV RT 5X Buffer	4.0
100mM DTT	1.0
RNaseOUT RNase Inhibitor	1.0
Superscript IV Reverse Transcriptase	1.0
Total	7.0

Master mix for RT reaction.

Note

The mastermix should be made up in the mastermix cabinet and added to the denatured RNA in the extraction and sample addition cabinet. Tubes should be wiped down when entering and leaving the mastermix cabinet.

Lot# _______________ Exp. Date _______________

1.6.

[ ] Add the above mastermix (7µL) to the annealed DNA (13µL) giving a total volume 20µL

1.7.

[ ] Cap the tube (or seal the plate), mix and then briefly centrifuge the contents.

1.8.

[ ] Preheat thermocycler to 42°C , with heated lid at 105°C

1.9.

[ ] Incubate sample using the following reverse transcription program:

A	B	C	D
Step	Temperature (°C)	Time	Cycle
Reverse Transcription	42	50:00	1
RT Inactivation	70	10:00	1
Cool	4	Hold	Hold

SARS-CoV-2 Reverse Transcription Program

PAUSE POINT cDNA can be stored at (same day) or (up to a week). 4°C (same day) or -20°C (up to a week).*

SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR

Tiled PCR Section Date/Initials:_________________

This section outlines the process for the tiled PCR approach from the ARTIC protocol.

Note

Primer pool sequences (v3) can be found here: https://github.com/joshquick/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019.tsv

If required, resuspend lyophilised primers at a concentration of 100 µM each.

Prepare the primer working solution diluting to 10micromolar (µM) using 0.1% volume TE buffer.

2.1.

[ ] Set up two individual reactions using primer pool 1 (set 1) and primer pool 2 (set 2) in 0.2mL PCR tubes according to the following table:

A	B	C	D	E
Reagent	Pool 1 (uL)	MM for N+2 samples	Pool 2 (uL)	MM for N+2 samples
Q5 Hot Start HiFi 2x MM	12.5		12.5
Primer pool at 10uM (1 or 2)	3.7		3.7
Nuclease-free water	6.3		6.3
Total	22.5		22.5

Master Mix for Tiled PCR

Lot# _______________ Exp. Date _______________

2.2.

[ ] Aliquot 22.5µL from the mastermix into 2 96-well PCR plates or 2 sets of PCR tubes.

2.3.

[ ] Add 2.5µL of sample cDNA (from step 1.9) to each pool giving a total volume 25µL and mix by pipetting. Spin briefly.

2.4.

[ ] Heat seal and place the plates onto a thermocycler and run the following program.

Important! Heat seal to minimise evaporation.

Note: Amplification should ideally be performed in a different lab to minimise the risk of contaminating other samples.

A	B	C	D
Step	Temperature	Time	Cycles
Initial Denaturation	98°C	0:30	1
Denaturation	98°C	0:15	35
Anneal and Extension	63°C	5:00	35
Cool	4°C	Hold	Hold

SARS-CoV-2 Tiled PCR Program

Note

Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35

Note

Pause point, Amplified cDNA can be stored at (overnight) or up to a week.

SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection

Section for Clean-Up and Size Selection Date/Initials:_________________

Reagent preparation:

Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.

Note

AMPure XP beads are needed in later steps. As the beads will need to equilibrate to room temperature before use, please consult steps 5.6 and 7.1 to ensure enough beads to cover all steps and save time. IMPORTANT: At all stages, ensure to homogenise beads before use.

Prepare the 80% volume ethanol (EtOH) using the following calculation:

0.360mL x (# Sample + 1: ________________) = ________ mL total volume (EtOH 100%)

mL total volume x 0.8 = ________ mL EtOH

Total volume _______mL - _______mL EtOH = ________mL H2O

3.1.

[ ] Combine the entire volumes of pool 1 and pool 2 PCR reactions (50µL in total) into one clean PCR plate (or PCR tubes set).

3.10.

[ ] Transfer the plate on the magnet and incubate for 0h 5m 0s at Room temperature

3.11.

[ ] Carefully transfer the supernatant (28μl) into a new plate, taking care not to disturb the bead pellet.

Note

PAUSE POINT Purified amplified cDNA can be stored at -20°C for several weeks prior to library preparation.

3.12.

[ ] Quantify the sample on Qubit fluorometer or similar instrument and store completed PCR amplified cDNA prep at -20°C

Note

Purified amplified cDNA is quantified with the use of the dsDNA HS Assay kit. 30 uL of samples should contain 50 ng to 1 ug of DNA (optimal 100-500 ng of DNA). If the DNA concentration at this step is less than ~3ng/uL, the sample did not amplify well and it could be under-represented in the final sequencing reaction.To streamline the workflow, the samples are not normalised but used as input for library preparation, the entire volume is used for the library preparation.To normalise, add enough DNA to reach a total of at least 100 ng** and add molecular grade water to bring the total volume to 30 µl.**NOTE: Preferred amount is 100 ng to 500 ng. Less than that can lead to under-representation of the sample in the final pool.

3.2.

[ ] Add 0.8X volume of SPRI beads per sample (40µL SPRI : 50µL amplified cDNA), mix well by pipetting.

Incubate 0h 10m 0s at Room temperature.

3.3.

[ ] Transfer the plate on the magnet and incubate for 0h 5m 0s at Room temperature.

3.4.

[ ] Keep the plate on the magnet and remove the superanatant by pipetting from the bottom.

Note

Keep the supernatant in case you have to go back for quality assessment. You may recycle one of the PCR plates used during the pool 1/pool 2 PCR stage to retain supernatant. Ensure to label plate correctly with step no. 3.4 and any unique identifiers for ease of finding later on.

3.5.

[ ] Wash the beads in the magnet with 180µL of freshly prepared 80 % volume EtOH without disturbing the pellet and incubate for 0h 0m 30s and remove the EtOH.

3.6.

[ ] Repeat previous step (total 2 washes).

3.7.

[ ] Spin down and place the tubes back on the magnet. Pipette off any residual ethanol with a P10 pipette and allow to dry for approximately 0h 10m 0s.

Note

Do not over-dry the beads. This may result in a lower recovery of DNA. Beads should appear dark brown and glossy. If they have become light brown or start to crack, this may be a sign they have become too dry. Dry beads may result in a lower recovery of DNA

3.8.

[ ] Remove the plate from the magnet and add 30µL of nuclease-free water, resuspend the beads pipetting up and down at least 10 times or vortex at 1800rpm,0h 0m 0s for 0h 1m 0s

3.9.

[ ] Incubate at room temperature for 0h 2m 0s

NEBNext library preparation protocol - Fragmentation/End prep

Note

At this point in the protocol, there are two options, enzymatic fragmentation and end repair. The method used is dependant upon preference and equipment/consumable/budgetary constraints in the lab. The enzymatic fragmentation (using NEBNext FS Library Prep Kit E7658) generates library inserts in the 150bp range compatible with 2 x 75 sequencing on illumina instruments. Follow steps 4.1 to 4.3 for this method. The end repaire method (using NEBNext Library prep kit E7650) repairs the ends of the ~400bp amplicons generated in the tiling PCR. These libraries will be ~400bp, compatible with 2 x 250 sequencing. Follow steps 4.4 to

This section is an adaptation protocol for FS DNA Library Prep Kit (E7805, E6177) with Inputs ≥ 100ng

Note

For inputs < 100 ng, size selection is not recommended. For 100 ng inputs, either the no size selection protocol or a size selection protocol can be followed.

4.1.

[ ] Prepare enzyme Master Mix using the following table:

A	B	C
Reagent	Volume (uL)	*(#samples+2)
NEBNext Ultra II FS Reaction Buffer	3.5 µl
NEBNext Ultra II FS Enzyme Mix	1 µl
Total Volume	4.5 µl

Note

Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice.

4.2.

[ ] Add 4.5µL of prepared mastermix (above) to each well. Add 13µL of purified DNA to the PCR tube or to the wells of the PCR plate.Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.

4.3.

[ ] In a Thermocycler, with the heated lid set to 75°C, run the following program:

A	B	C
Step	Temp	Time
1	37°C	30 min
2	65°C	30 min
Hold	4°C	Hold

Note

OPTIMIZATIONFragmentation occurs during the 37°C incubation step. Use the chart below to determine the incubation time required to generate the desired fragment sizes. Incubation time may need to be optimized for individual samples. Run the fragmented suspension on Bioanalyzer to visualize the size distribution. FRAGMENTATION SIZE INCUBATION @ 37°C OPTIMIZATION 100 bp-250 bp 30 min 30-40 min 150 bp-350 bp 20 min 20-30 min 200 bp-450 bp 15 min 15-20 min 300 bp-700 bp 10 min 5-15 min 500 bp-1 kb 5 min 5-10 min https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bgttjwnn?step=26

Note

If necessary, samples can be stored at –-20°C , however, a slight loss in yield (~20%) may be observed. It is recommend continuing with adaptor ligation before stopping.Continue with this protocol from step 5.

4.4.

Steps 4.1 to 4.3 detailed enzymatic fragmentation. The following steps (4.4 to 4.6) detail the end repair option.

If you have carried out steps 4.1 to 4.3, this protocol continues from step 5 *

[ ] Prepare the following mastermix in a sterile nuclease-free tube:

A	B
Component	Volume
NEBNext Ultra II End Prep Enzyme Mix	1.5 µl
NEBNext Ultra II End Prep Reaction Buffer	3.5 µl

Total Volume	5 µl

4.5.

[ ] Add 5µL of mastermix (above) to each well. Add 25µL of purified DNA to the PCR tube or to the wells of the PCR plate.Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.

4.6.

[ ] In a thermocycler, with the heated lid set to 75°C, run the following program:

A	B
Temperature	Time
20 °C	30 min
65 °C	30 min
4 °C	∞

Note

If necessary, samples can be stored at –-20°C , however, a slight loss in yield (~20%) may be observed. It is recommend continuing with adaptor ligation before stopping.

NEBNext library preparation protocol - Adapter ligation

[ ] Add the following components directly to the FS Reaction Mixture:

A	B
Component	Volume
FS Reaction Mixture (Step 4.3) or End Prep Reaction Mixture (step 4.6)	17.5 µl/ 30 µl
NEBNext Ultra II Ligation Master Mix	15 µl
NEBNext Adaptor for Illumina	1.25µl
Total Volume	33.75 µl/ 46.25 µl

Note

It is not recommended to add adaptor to a premix in the Adaptor Ligation Step.For this reason, add Ligation Master mix to each well/PCR tube, then add 1.25μl adapter at the end. Seal plate, vortex for 0h 0m 10s & spin briefly

5.1.

[ ] Incubate at 20°C for 0h 15m 0s in a thermocycler with the heated lid off .

Cleanup of Adaptor-ligated DNA

5.10.

[ ] Wash the beads adding 200µL of freshly prepared 80% ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant.

Be careful not to disturb the beads that contain DNA targets.

5.11.

[ ] Repeat Step 5.10 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

5.12.

[ ] Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Note

Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

5.13.

[ ] Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 10µL 0.1% volume TE (dilute 1X TE Buffer 1:10 in water).

5.14.

[ ] Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 0h 2m 0s at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

5.15.

[ ] Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 7.5µL to a new PCR tube.

Note

Samples can be stored at -20°C

5.2.

[ ] Add 1.5µL µl of USER Enzyme to the ligation mixture from Step 5.1. Vortex for 0h 0m 10s & spin briefly.

5.3.

[ ] Mix well and incubate in thermocycler at 37°C for 0h 15m 0s with the heated lid set to ≥ 47°C

Note

Samples can be stored overnight at -20°C

5.4.

Note

The volumes of Ampure XP Breads will vary depend on fragmentation method used in section 4.

5.5.

[ ] Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.

5.6.

[ ] Add 28µL (FS fragmentation) or 43µL(end repair) of the Ampure XP Beads to the ligation reaction mixture and mix well by pipetting up and down, or vortex. Spin briefly.

5.7.

[ ] Incubate at room temperature for 0h 5m 0s

5.8.

[ ] Place the plate on magnetic block for 0h 5m 0s

5.9.

[ ] Carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets

NEBNext library preparation protocol - PCR Enrichment of Adapter-ligated DNA

[ ] Add the following reagents to each well from step 5.15

A	B
Component	Volume
Adaptor Ligated DNA Fragments (Step 5.15)	7.5 µl
NEBNext Ultra II Q5 Master Mix	12.5 µl
Index Primer/i7 Primer	2.5 µl
Universal PCR Primer/i5 Primer	2.5 µl
Total Volume	25 µl

Note

Ensure to take note of what index set (1 or 2) is used and their sequence numbers,

Index set no. _______________

Index Range (A) _______________ Index Range (B) _______________

6.1.

[ ] Set a 100 µl or 200 µl pipette to 40 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

6.2.

[ ] Place the tube/plate on a thermocycler with the heated lid set to 105°C and perform PCR amplification using the following PCR cycling conditions:

A	B	C	D
CYCLE STEP	TEMP	TIME	CYCLES
Initial Denaturation	98°C	30 seconds	1
Denaturation	98°C	10 seconds	5*
Annealing/Extension	65°C	75 seconds
Final Extension	65°C	5 minutes	1
Hold	4°C	∞

Note

*Cycle number was determined by size of input DNA ~100ng is 4-5 cycles.

NEBNext library preparation protocol - Clean up of PCR reaction

Allow the Ampure XP beads to warm to room temperature for at least 30 minutes before use.

[ ] Vortex SPRIselect to resuspend.

7.1.

[ ] Add 22.5µL (0.9X) resuspended beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

7.10.

[ ] Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15µL to a new PCR tube and store at -20°C.

7.2.

[ ] Incubate samples on bench top for at least 0h 5m 0s at Room temperature

7.3.

[ ] Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

7.4.

[ ] After 0h 5m 0s (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard the beads).

7.5.

[ ] Add 200µL of 80% volume freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 0h 0m 30s , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

7.6.

[ ] Repeat Step 7.5. once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

7.7.

[ ] Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Note

7.8.

[ ] Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17µL of 0.1% (v/v) TE (dilute 1X TE Buffer 1:10 in water).

7.9.

Assess Library quality

Set up dilutions and standards as laid out in the kit protocol for dsDNA high sensitivity kit.

Record Qubit readings before normalization.

Note

In this protocol 2µL of library (198µL buffer)

8.1.

[ ] Run Samples on Agilent Bioanalyser or Agilent Tapestation to check that the library shows a narrow distribution with an expected peak size based on fragmentation time and size selection. Record the the average peak bp size.

Note

Tape station D1000 HS 2µL of library in 2µL buffer (ladder 2µL in 2µL buffer for each cartridge)

8.2.

[ ] Calculate the dilutions required to normalise each sample to a 4nM concentration using the following formula:

Note

Note: If a peak ~80 bp (primers) or 128 bp (adaptor-dimer) is visible in the Bioanalyzer trace, bring up the sample volume (from Step 2.5.11.) to 50 µl with 0.1X TE Buffer and repeat the Cleanup of PCR Reaction in Section 2.5.

[ ] Run Samples on a bioanalyser or tapestation and check that the library shows a narrow distribution with an expected peak size based on fragmentation time and size selection. Record the the average peak bp size

Note

Calculate the molar concentration of each library to be diluted using average size from the TapeStation and mass from Qubit, using the following equation: (????????????? (??⁄??)) / (660 ?⁄??? × ??? ??????? ???????? ????) × 10^6 ??/? = ????????????? (??)Make a 4nM dilution of each library

MiSeq Sequencing

10.

Pooling and Library Denaturation Date/Initials:_________________

This section demonstrates how to generate a pooled library for V2 reagents on the MiSeq.

Note

Thaw the MiSeq reagents overnight or in aRoom temperature waterbath.Remove HT1 from freezer and thaw at Room temperature. Store at 2°C to8°C until you are ready to dilute denatured libraries.

Note

Label 3 eppendorfs for: (1) the pooled library(2) denaturation of library(3) 0.2N NaOHMake a fresh dilution of 0.2N of NaOH by combining the following volumes in a microcentrifuge tube: 800µL laboratory-grade water and 200µL stock 1.0nanomolar (nM) NaOH

10.1.

[ ] Pool 5µL of each normalised sample into an eppendorf tube. This will be (1) pooled library.

10.10.

[ ] Set aside on ice until you are ready to load it onto the reagent cartridge.

10.11.

[ ] Mix reagents of the MiSeq cartridge thoroughly by inverting several times.

[ ] Using a fresh 1000 µL pipette tip, transfer the denatured and library (with PhiX spiked) into position 17.

10.12.

[ ] Load the sample sheet and reagents according to onscreen instructions in the MiSeq Control software.

10.2.

[ ] Combine the following volumes in a microcentrifuge tube (2):

5µL 4nM pooled library and 5µL of 0.2 N NaOH.

[ ] Vortex briefly and then centrifuge at 280 x g for 1 minute.

[ ] Incubate at room temperature for 0h 5m 0s

10.3.

[ ] Add 990µL of pre-chilled HT1 to the tube containing the denatured library (2). The result is 1 mL of a 20 pM denatured library.

10.4.

[ ] Dilute the 20 pM library to the desired concentration, see table below:

A	B	C	D	E	F	G
Concentration	6 pM	8 pM	10 pM	12 pM	15 pM	20 pM
20 pM library	180 uL	240 uL	300 uL	360 uL	450 uL	600 uL
Pre-chilled HT1	420 uL	360 uL	300 uL	240 uL	150 uL	0 uL

Note

We recommend diluting the library to 10pM for optimal cluster density during Miseq runs with V2 reagents.

[ ] Invert to mix and then pulse centrifuge

Note

The following steps 10.5 to 10.7 can be carried out ahead of time and PhiX library can be stored at -20°C for a number of weeks

10.5.

[ ] Dilute stock PhiX to 4nM by combining:

2µLof 10nanomolar (nM) PhiX library
3µLof 10millimolar (mM) Tris-Cl, pH 8.5 with 0.1% Tween 20

10.6.

Denature the PhiX control by adding the following volumes in a microcentrifuge tube:

5µL of 4nanomolar (nM) PhiX library
5µL of 0.2nanomolar (nM) NaOH

Note

Remaining 4nanomolar (nM) PhiX can be frozen and reused

10.7.

[ ] Vortex briefly to mix and centrifuge at 280x g for0h 1m 0s.

[ ] Incubate at8Room temperature for 0h 5m 0s

10.8.

[ ] Dilute denatured PhiX library to 20 pM by adding 990 uL pre-chilled HT1 to the PhiX tube. Invert to mix.

Note

If using a MiSeq reagent kit v2, dilute 20 pM PhiX library to 12.5 pM by adding the following volumes in a microcentrifuge tube: - 375 µL 20 pM denatured PhiX library - 225 µL pre-chilled HT1

10.9.

[ ] Combine library and PhiX control according to the table below:

A	B
Denatured and diluted PhiX (12.5pM)	30 µl
Denatured and diluted library (10 pM)	570 µl

SARS-CoV-2 Whole Genome Sequencing on Illumina

Disclaimer

Abstract

Steps

SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription

SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR

SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection

NEBNext library preparation protocol - Fragmentation/End prep

NEBNext library preparation protocol - Adapter ligation

Cleanup of Adaptor-ligated DNA

NEBNext library preparation protocol - PCR Enrichment of Adapter-ligated DNA

NEBNext library preparation protocol - Clean up of PCR reaction

Assess Library quality

MiSeq Sequencing

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