TITLE: NEBnext library construction and sequencing for SARS-CoV-2: Adapting  COVID-19 ARTIC protocol
AUTHORS: Jennifer Giandhari, Sureshnee Pillay, Houriiyah Tegally, Eduan Wilkinson, Benjamin Chimukangara, Richard Lessells, Yunus Moosa, Inbal Gazy, Maryam Fish, Lavanya Singh, Khulekani Sedwell Khanyile, Vagner Fonseca, Marta Giovanetti, Luiz Carols Alcantara, Tulio de Oliveira

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently producing amplicons tiling the viral genome sequencing. It uses the V3 nCov-2019 primers  from the ARTIC network. This is followed by library construction using Nextera Flex, which we found to save 9h of hands on time as compared with original protocol that uses TruSeq for library contstruction. It also describes the pooling of samples and quantitation, prior to sequencing on the Illumina Miseq. </div><div class = "text-block">It is adapted from the nCov-2019 sequencing protocol from Quick and colleagues, which can be found here: </div></div>

[STEPS]
?. [cDNA]
Prepare the cDNA mastermix in the pre-PCR clean room. The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
?. [cDNA]
Mix the following components in a labeled 1.5ml Component:  AB1ComponentVolume (ul)250μM Random Hexamers 11310mM dNTPs mix (10mM each) 114Template RNA115Total13
AB1ComponentVolume (ul)250μM Random Hexamers 11310mM dNTPs mix (10mM each) 114Template RNA115Total13
?. [cDNA]
Add  to a labeled 1.5ml eppendorf tube.
[50μM Random Hexamers 1]
?. [cDNA]
Add .
[10mM dNTPs mix (10mM each) 1]
?. [cDNA]
Add .
[Template RNA]
The total volume in the tube should now be  .
?. [cDNA]
Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.
?. [cDNA]
Aliquot the mastermix in labelled PCR strip tubes.
PCR master mixes (shown in Tables 1 and 3) can be prepared at the same time, in the pre-PCR area before starting amplifications.
?. [cDNA]
Incubate the reaction as follows in a thermal cycler.  AB1Temperature (°C)Time2655 minutes341 minute
AB1Temperature (°C)Time2655 minutes341 minute
?. [cDNA]
Spin down the tubes with the RNA and primers to get all liquid to the bottom.
?. [cDNA]
Prepare the following mastermix in the clean mastermix room.Mix the following components in a labeled 1.5ml eppendorf tube:  AB1ComponentVolume (μl)2SSIV Buffer43100mM DTT14RNaseOUT RNase Inhibitor15SSIV Reverse Transcriptase16Total7
AB1ComponentVolume (μl)2SSIV Buffer43100mM DTT14RNaseOUT RNase Inhibitor15SSIV Reverse Transcriptase16Total7
?. [cDNA]
Add  to a labeled 1.5ml eppendorf tube.
[SSIV Buffer]
?. [cDNA]
Add .
[100mM DTT]
?. [cDNA]
Add .
[RNaseOUT RNase Inhibitor]
?. [cDNA]
Add .
[SSIV Reverse Transcriptase]
The total volume should now be  .
?. [cDNA]
The mastermix must be added to the  for a .
[denatured RNA]
[total volume]
?. [cDNA]
Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.
?. [cDNA]
Incubate the reaction as follows in a thermal cycler.  AB1Temperature (°C)Time24250 minutes37010 minutes45Hold
AB1Temperature (°C)Time24250 minutes37010 minutes45Hold
?. [Primer Pool Preparation]
Primers must be diluted and pooled using nuclease free water in a clean mastermix hood. The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
?. [Primer Pool Preparation]
If required, resuspend lyophilised primers at a concentration of  each.
2019- nCoV primers for this protocol were designed using Primal Scheme to generate overlapping 400 nucleotide amplicons.
?. [Primer Pool Preparation]
To generate , add  (named pool 1 or pool 2) to a 1.5ml eppendorf tube labeled either “Pool 1 (100μM)” or “Pool 2 (100μM)”.
[primer pool stocks]
[of each primer pair]
Total volume will be   for Pool 1 (100uM) and   for Pool 2 (100uM). These are now   stocks of each primer pool.
?. [Primer Pool Preparation]
Dilute the 100μM primer pool 1:10 in molecular grade water, to generate .
[primer stocks]
It is recommended that multiple aliquots of each primer pool are made in case of degradation or contamination.
?. [Primer Pool Preparation]
Primers need to be used at a final concentration of  . In this case both pools have 98 primers in, so the requirement is   per  .
?. [Tiling PCR]
Prepare the PCR mastermix in the clean mastermix room.
?. [Tiling PCR]
The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
?. [Tiling PCR]
A mastermix for each pool must be made up in the mastermix hood.Mix the following components in a labeled 1.5ml eppendorf tube:  ABC1ComponentPool 1 volumes (μl)Pool 2 volumes (μl)25X Q5 Reaction Buffer55310mM dNTPs0.50.54Q5 Hot Start DNA Polymerase0.250.255Primer Pool 1 or 2 (10μM)3.63.66Nuclease-free water10.6510.657Total2020
ABC1ComponentPool 1 volumes (μl)Pool 2 volumes (μl)25X Q5 Reaction Buffer55310mM dNTPs0.50.54Q5 Hot Start DNA Polymerase0.250.255Primer Pool 1 or 2 (10μM)3.63.66Nuclease-free water10.6510.657Total2020
?. [Tiling PCR]
Add  to a labeled 1.5ml eppendorf tube.
[5X Q5 Reaction Buffer]
?. [Tiling PCR]
Add .
[10mM dNTPs]
?. [Tiling PCR]
Add .
[Q5 Hot Start DNA Polymerase]
?. [Tiling PCR]
Add .
[Primer Pool 1 or 2 (10μM)]
?. [Tiling PCR]
Add .
[Nuclease-free water]
The total volume should now be  .
?. [Tiling PCR]
Aliquot the mastermix in labelled PCR strip tubes.
?. [Tiling PCR]
Add  under the extraction hood or general lab hood, which has been decontaminated using with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
[of cDNA]
?. [Tiling PCR]
Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.
?. [Tiling PCR]
Incubate the reaction as follows in a thermal cycler.  ABCD1StepTemperature (°C)TimeCycles2Heat Activation9830 seconds13Denaturation9815 seconds354Annealing655 minutes5Hold4∞
ABCD1StepTemperature (°C)TimeCycles2Heat Activation9830 seconds13Denaturation9815 seconds354Annealing655 minutes5Hold4∞
?. [PCR Clean-up]
Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into a single 1.5 ml eppendorf tube.
?. [PCR Clean-up]
Vortex Ampure beads thoroughly to ensure they are well resuspended; the solution should be a homogenous brown colour.
?. [PCR Clean-up]
Add an equal volume (1:1) of Ampure beads to the pooled sample tube and mix gently by either flicking or pipetting.
For example, add   to a  .
?. [PCR Clean-up]
Pulse centrifuge to collect all liquid at the bottom of the tube.
?. [PCR Clean-up]
Incubate for  at .
0 Room temperature
?. [PCR Clean-up]
Place on magnetic rack and incubate for  or until the beads have pelleted and the supernatant is completely clear.
?. [PCR Clean-up]
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
?. [PCR Clean-up]
Add  (at ) to the pellet.
[of freshly prepared 70% ethanol]
0 Room temperature
?. [PCR Clean-up]
Carefully remove and discard ethanol, being careful not to touch the bead pellet.
?. [PCR Clean-up]
Add  (at ) to the pellet.
[of freshly prepared 70% ethanol]
0 Room temperature
?. [PCR Clean-up]
Carefully remove and discard ethanol, being careful not to touch the bead pellet.
?. [PCR Clean-up]
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
?. [PCR Clean-up]
With the tube lid open incubate for  or until the pellet loses its shine.
If the pellet dries completely it will crack and become difficult to resuspend
?. [PCR Clean-up]
Resuspend pellet in , mix gently by either flicking or pipetting and incubate for .
[Elution Buffer (EB)]
?. [PCR Clean-up]
Place on magnetic stand and transfer sample to a clean 1.5mL eppendorf tube ensuring no beads are transferred into this tube.
?. [PCR Clean-up]
***Sample concentration can be determined using the Qubit and the size of amplicons can be visualized using the LabChip Fragment Analyzer.
The expected amplicon size is 400bp.
?. [Library Preparation End Repair]
Prepare the end repair master mix.Multiply each volume by the number of samples being processed  AB1ComponentVolume (μl) per sample2NEBNext Ultra II End Prep Enzyme Mix (green top)33NEBNext Ultra II End Prep Reaction Buffer (green top)74Total10
AB1ComponentVolume (μl) per sample2NEBNext Ultra II End Prep Enzyme Mix (green top)33NEBNext Ultra II End Prep Reaction Buffer (green top)74Total10
?. [Library Preparation End Repair]
Please scale this step as needed. For :----
50
[NEBNext Ultra II End Prep Enzyme Mix (green top)]
[NEBNext Ultra II End Prep Reaction Buffer (green top)]
[Total]
?. [Library Preparation End Repair]
Seal the plate with a plate sealer, place on the preprogrammed thermal cycler, and run the end repair program.  AB1Temperature (°C)Time22030 minutes36530 minutes410Hold
AB1Temperature (°C)Time22030 minutes36530 minutes410Hold
If necessary, samples can be stored at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.
?. [Adapter Ligation]
Make up the following Mastermix and add 31μl to the respective wells.
?. [PCR Enrichment]
Combine the following volumes to prepare the PCR master mix. Multiply each volume by the number of samples being processed.  AB1ComponentVolume (μl) per sample2NEBNext Ultra II Q5 Master Mix (blue top)253Universal PCR primer (blue top)54Total30
AB1ComponentVolume (μl) per sample2NEBNext Ultra II Q5 Master Mix (blue top)253Universal PCR primer (blue top)54Total30
The PCR will take approximately 30 minutes
?. [PCR Enrichment]
ABC1Temperature (°C)TimeCycles2983 minutes139810 seconds846030 seconds56545 seconds6655 minutes174Hold
ABC1Temperature (°C)TimeCycles2983 minutes139810 seconds846030 seconds56545 seconds6655 minutes174Hold
?. [Clean-up of Enrichment PCR]
If using AMPure XP Beads, allow the beads to warm to room temperature for at least 30 minutes before use
?. [Normalization of DNA]
Calculate appropriate amount of diluent in 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
?. [Normalization of DNA]
Pipette mix 5 times.
?. [Normalization of DNA]
Use a multi-channel pipette to transfer  to an 8 strip-tube and spin briefly.
[of the diluted sample library]
?. [Normalization of DNA]
Pool the library samples from the 8-strip tubes to a labelled Pooled Amplicon Library (PAL) 2ml eppendorf tube.
?. [Normalization of DNA]
Proceed to library denaturation.
?. [Library Denaturation]
Remove the tube of HT1 (Hybridization Buffer) from the freezer (-15°C to -25°C) and set aside at  to thaw.
-15 Room temperature
?. [Library Denaturation]
When thawed, store at  to  until you are ready to dilute denatured libraries.
2 °C
8 °C
?. [Library Denaturation]
Prepare 500μl of 0.2 N NaOH by combining the following volumes in a 1.5ml microcentrifuge tube:  and .Refer to the formula below:1M=1N10N(x)=(0.2)(500)x =  +
[laboratory-grade water]
[Stock 1.0 N NaOH]
[NaOH]
[laboratory-grade water]
A fresh dilution of 0.2N NaOH is required for the denaturation process in preparing sample DNA and a PhiX control.
?. [Library Denaturation]
Invert the tube several times to mix.
?. [Library Denaturation]
Combine the following volumes of pooled sample DNA and freshly diluted 0.2 N NaOH in a micro-centrifuge tube, by adding  to .
[of 4nM sample DNA]
[of 4nM sample DNA]
?. [Library Denaturation]
Discard the remaining dilution of 0.2 N NaOH or set aside to prepare a PhiX control within the next .
?. [Library Denaturation]
Vortex briefly to mix the sample solution, and then centrifuge the sample solution to .
Centrifuge: 280 34, 1 min
?. [Library Denaturation]
Incubate for  at  to denature the DNA into single strands.
8 Room temperature
?. [Library Denaturation]
Add  to .
[of 4nM sample DNA]
[of pre-chilled HT1]
The result is a 20pM denatured library in 1 mM NaOH.
?. [Library Denaturation]
Place the denatured DNA  or at  until you are ready to proceed to the final dilution.
on ice
4 °C
?. [Dilution of Denatured Library]
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 5% PhiX):  ABCD1Final Concentration20pM denatured DNA5% PhiXPre-chilled HT1212pM356.4ul3.6ul240ul
ABCD1Final Concentration20pM denatured DNA5% PhiXPre-chilled HT1212pM356.4ul3.6ul240ul
?. [Dilution of Denatured Library]
Invert several times to mix and then pulse centrifuge.
?. [Dilution of Denatured Library]
To dilute PhiX to 4nM concentration, combine the following volumes in a microcentrifuge tube:• • ,  with
[of 10nM PhiX library]
[of 10mM Tris-Cl]
[Tween 20]
If not prepared within the last 12 hours, prepare a fresh dilution of 0.2 N NaOH.
?. [Dilution of Denatured Library]
Combine the following volumes in a micro-centrifuge tube:• •
[of 4 nM PhiX library]
[of 0.2 N NaOH]
?. [Dilution of Denatured Library]
Vortex briefly to mix.
?. [Dilution of Denatured Library]
Centrifuge at .
Centrifuge: 280 34, 1 min
?. [Dilution of Denatured Library]
Incubate at  for .
8 Room temperature
?. [Dilution of Denatured Library]
Dilute denatured PhiX to 20pM by adding pre-chilled HT1 to the denatured PhiX library as follows: • •
[denatured PhiX library]
[pre-chilled HT1]
?. [Dilution of Denatured Library]
Invert to mix.
?. [Dilution of Denatured Library]
Combine Library and PhiX Control.
?. [Dilution of Denatured Library]
Mix this solution well and briefly centrifuge.
?. [Dilution of Denatured Library]
Keep  or at  until it is ready to be loaded onto the MiSeq reagent cartridge.
on ice
4 °C
?. [Library Preparation End Repair]
For a 400bp insert, use 200ng input DNA. Input amounts lower than those specified results in low yield and increased duplicates
Starting Material: Cleaned-up DNA diluted to 1 – 5 ng/μl, in at least 50 μl volume.
?. [Library Preparation End Repair]
Add 50μl of DNA to the respective wells, for a total reaction volume of 60 μl.
?. [Library Preparation End Repair]
Mix well on a vortex mixer or by pipetting up and down 10 times.
?. [Library Preparation End Repair]
Perform a quick spin to collect all liquid from the sides of the tube.
It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
?. [Library Preparation End Repair]
Place in a thermocycler, with the heated lid set to ≥75°C, and run the following program:
?. [Adapter Ligation]
Add 35μl of the End Prep DNA to the respective wells.
?. [Adapter Ligation]
Add 2.5μl of NEBNext Adpaters for Illumina to the respective wells.
?. [Adapter Ligation]
Mix well on a vortex mixer or by pipetting up and down 10 times.
?. [Adapter Ligation]
Perform a quick spin to collect all liquid from the sides of the tube. Incubate at 20°C for 15 minutes in a thermocycler with the heated lid open.
The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. We do not recommend adding adaptor to a premix in the Adaptor Ligation Step.
The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance). 
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							   Note: Samples can be stored overnight at –20°C.
?. [Clean-up of Adapter Ligated DNA]
Allow AMPure XP Beads to warm to room temperature for at least 30 minutes before use.
?. [Clean-up of Adapter Ligated DNA]
Vortex AMPure Beads thoroughly to resuspend.
?. [Clean-up of Adapter Ligated DNA]
Add 57μl (~0.8X) of resuspended beads to the adaptor ligation reaction.
?. [Clean-up of Adapter Ligated DNA]
Mix well by vortexing for 3 – 5 seconds or by pipetting up and down 10 times.
?. [Clean-up of Adapter Ligated DNA]
Centrifuge briefly. Be sure to stop the centrifugation before the beads start to settle out.
?. [Clean-up of Adapter Ligated DNA]
Incubate samples on bench top for at least 5 minutes at room temperature.
?. [Clean-up of Adapter Ligated DNA]
Place the tube/plate on an appropriate magnetic stand for 5 minutes to separate the beads from the supernatant.
?. [Clean-up of Adapter Ligated DNA]
Carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.
?. [Clean-up of Adapter Ligated DNA]
Incubate at room temperature for 30 seconds.
?. [Clean-up of Adapter Ligated DNA]
Carefully remove and discard the supernatant without disturbing the pellet.
?. [Clean-up of Adapter Ligated DNA]
Add 200μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. 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.
?. [Clean-up of Adapter Ligated DNA]
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Do not over-dry the beads. This may result in lower recovery of DNA target. 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.
?. [Clean-up of Adapter Ligated DNA]
Remove the tube/plate from the magnetic stand.
?. [Clean-up of Adapter Ligated DNA]
Add 17μl of nuclease free water to elute the DNA target from the beads.
?. [Clean-up of Adapter Ligated DNA]
Mix well on a vortex mixer or by pipetting up and down 10 times.
?. [Clean-up of Adapter Ligated DNA]
Incubate at room temperature for at 2 minutes.
?. [Clean-up of Adapter Ligated DNA]
Quickly spin to collect the liquid from the sides of the tube or plate wells.
?. [Clean-up of Adapter Ligated DNA]
Place the tubes/ plate on the magnetic stand and incubate for at least 5 minutes at room temperature.
?. [Clean-up of Adapter Ligated DNA]
Transfer 15μl (i.e. 7.5μl twice) of the supernatant to a new tubes/ plate for amplification.
Samples can be stored at –20°C.
?. [PCR Enrichment]
Add 30μl of the master mix into newly labelled tubes/ plate.
?. [PCR Enrichment]
Add 15.0μl of adaptor-ligated DNA to the respective wells.
?. [PCR Enrichment]
Mix well on a vortex mixer or by pipetting up and down 10 times.
?. [PCR Enrichment]
Perform a quick spin to collect all liquid from the sides of the tube.
?. [PCR Enrichment]
Place the tubes/plate on a thermocycler and perform PCR using the following conditions:
?. [Clean-up of Enrichment PCR]
Vortex AMPure Beads thoroughly to resuspend.
?. [Clean-up of Enrichment PCR]
Add 45μl (0.9X) resuspended AMPure beads to the PCR reaction.
?. [Clean-up of Enrichment PCR]
Mix well by vortexing for 3 – 5 seconds or by pipetting up and down 10 times.
?. [Clean-up of Enrichment PCR]
Centrifuge very briefly. Be sure to stop the centrifugation before the beads start to settle out.
?. [Clean-up of Enrichment PCR]
Incubate samples on bench top for at least 5 minutes at room temperature.
?. [Clean-up of Enrichment PCR]
Place the tube/plate on an appropriate magnetic stand for 5 minutes to separate the beads from the supernatant.
?. [Clean-up of Enrichment PCR]
Carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.
?. [Clean-up of Enrichment PCR]
Add 200μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand.
?. [Clean-up of Enrichment PCR]
Incubate at room temperature for 30 seconds.
?. [Clean-up of Enrichment PCR]
Carefully remove and discard the supernatant without disturbing the pellet.
?. [Clean-up of Enrichment PCR]
Add 200μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. 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.
?. [Clean-up of Enrichment PCR]
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Do not over-dry the beads. This may result in lower recovery of DNA target. 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.
?. [Clean-up of Enrichment PCR]
Remove the tube/plate from the magnetic stand.
?. [Clean-up of Enrichment PCR]
Add 33μl of nuclease free water to elute the DNA target from the beads.
?. [Clean-up of Enrichment PCR]
Mix well on a vortex mixer or by pipetting up and down 10 times.
?. [Clean-up of Enrichment PCR]
Quickly spin to collect the liquid from the sides of the tube or plate wells.
?. [Clean-up of Enrichment PCR]
Incubate the tubes/ plate for 2 minutes at room temperature.
?. [Clean-up of Enrichment PCR]
Place the tubes/ plate on the magnetic stand and incubate for at least 5 minutes.
?. [Clean-up of Enrichment PCR]
Transfer 30μl (i.e. 15.5μl twice) of the supernatant to a new tubes/ plate.
?. [Clean-up of Enrichment PCR]
Assess the concentration of the libraries using a Qubit fluorometer.
?. [Clean-up of Enrichment PCR]
Assess the library fragments using the LabChip GX Touch.
Samples can be stored at –20°C after clean-up.
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