TITLE: nCoV-2019 sequencing protocol v3 (LoCost) - InvitaeSF v2.0
AUTHORS: Amanda Kahn-Kirby, Lisa Cunden, Bianca Miani [Invitae

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Amplicon sequencing protocol for SARS-CoV-2 v3 (LoCost), modified for automation and clean yield.  Thank you Josh Quick and colleagues for publishing and revising the protocol on which this version is based.</div><div class = "text-block">We thank the ARTIC network, Oxford Nanopore Technologies, New England Biolabs, BCCDC, COG-UK, CanCOGen, other California SARS-CoV-2 sequencing labs, and protocols.io commenters for their assistance developing this protocol and sharing their work globally.  We hope this work is useful to other investigators a) dealing with low yield or b) looking to streamline the protocol through automation and miniaturization.</div><div class = "text-block">Changes to the Quick lab v3 version:</div><div class = "text-block">-Batch size 24 or 48 wells per run with EXP-NBD196 native barcode kit</div><div class = "text-block">-Use of LunaScript RT SuperMix</div><div class = "text-block">-10uL ARTIC reactions</div><div class = "text-block">-Post-PCR SPRI to improve barcode ligation efficiency</div><div class = "text-block">-Post-adapter SPRI stringency increased to decrease adapter reads</div><div class = "text-block">- Calculations are for 3-col or 6-col preparation on an Agilent Bravo</div></div>

[STEPS]
?. [cDNA preparation]
Stamp input plate to a 3-col or 6-col pre-aliquoted RT master mix plate using the Bravo protocol.  AB1ComponentVolume2LunaScript RT SuperMix (5X)   2 µL3Normalized Template RNA  8 µL4Total10 µL
AB1ComponentVolume2LunaScript RT SuperMix (5X)   2 µL3Normalized Template RNA  8 µL4Total10 µL
Viral RNA input from a clinical sample should be between Ct 18-35. If Ct is between 12-15, then dilute the sample 100-fold in water, if between 15-18 then dilute 10-fold in water. This will reduce the likelihood of PCR-inhibition.  The cherrypicking protocol does this step automatically. RNA samples should be handled in the Pre-PCR cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
To prevent pre-PCR contamination the mastermix should be pre-aliquoted in a sterile cabinet and stored at -20.
?. [cDNA preparation]
Incubate the reaction as follows: for  for  for Hold at
25 °C
55 °C
95 °C
4 °C
?. [Multiplex PCR]
Add  cDNA to each of the PCR reactions by Bravo stamp, followed by pipet-mix. Seal and pulse spin the plate to collect liquid at the bottom of the tube.
1.25 µl
?. [Multiplex PCR]
Set-up the following program on the thermal cycler:  Step                        Temperature     Time                    CyclesHeat Activation         1Denaturation              30Annealing                   30Hold                                   Indefinite              1
98 °C
98 °C
63 °C
4 °C
Thermocycler calibration can vary instrument to instrument. We saw amplicon 64 dropout so decreased the annealing/extension temperature to  .
?. [Native barcoding]
Barcode the amplicon pools using the one-pot native barcoding approach. 3-col and 6-col plates are used as input for the protocol. [Not modifying the One-Pot embedded protocol, but see the substeps below for automation-specific calculations at each step.
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?. [Native barcoding]
Quantify  of the barcoded amplicons using the Qubit hsDNA assay. Concentration will vary depending on number and Ct of samples and but you need about   total at this stage to achieve maximum run yield.
1 µl
30 ng
?. [Native barcoding]
Set up the following AMII adapter ligation and clean-up with SFB, but note that the SPRI ratio for the final clean should be 0.9X to minimize adapter reads. Elution is with Oxford Buffer EB, not Qiagen.  There is no Ethanol clean-up in the final SPRI.
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?. [Native barcoding]
Quantify  of the final library using the Qubit hsDNA assay. Concentration will vary depending on number and Ct of samples but  final library is usually required to achieve maximum run yield.
1 µl
15 ng
Final library can be now be stored in   Tris    at    for up to a week if needed otherwise proceed directly to MinION sequencing.
?. [MinION sequencing]
Prime the flowcell and load  sequencing library onto the flowcell.
15 ng
From experience we know    is optimum loading input for short amplicons. Speed drop during the run indicates excessive library was loaded. Low run yield
?. [MinION sequencing]
Start the sequencing run using MinKNOW.
If using Live basecalling ensure to turn on double-ended barcoding in the basecalling settings.
?. [cDNA preparation]
Input to the protocol is 22 or 46 RNA samples plus 1 positive control and one randomly-located negative control of nuclease-free water per library.  (Use Cherrypicking program to prepare the input plate to this protocol). If previously frozen, mix by briefly vortexing and pulse spin to collect liquid. Keep samples on ice at all times.
The recommended positive control is Twist Control 2 diluted in pooled SARS-CoV-2 negative RNA extract from nasal swab testing, 10K copies per reaction.  It is in a constant plate position for easy monitoring.
?. [Primer pool preparation (only for a fresh IDT box)]
Dilute IDT stock   pools 1:10 in molecular grade water, to generate  primer stocks.  ( ship volume +  molecular-grade water.    Aliquot into convenient-sized labeled aliquots ( ) and store at -20.
180 µl
1620 µl
65 µl
Primers are used at a final concentration of  per primer. In this case V3 pools have 110 primers in pool 1 and 108 primers in pool 2. so the requirement is ~  primer pool ( ) per   reaction.
?. [Multiplex PCR]
Set up the master mixes for two half-scale PCR reaction pools as follows. Use 1 aliquot set of pre-diluted  primers for a 3-col prep, and 2 sets for a 6-col prep.Gently mix the MM tube by pipetting/flicking and pulse spin the tube to collect liquid at the bottom of the tube.  Allocate to a single column of the Pre-PCR reagent plate, then distribute using the Bravo.  There will be residual dead volume in the MM tube and in the distribution plate (15% overage).Pool 1 setupComponentReaction 1 (each)Reaction 1 (3-col)Reaction 1 (6-col)Q5 Hot Start High-Fidelity 2X Master Mix6.25 µL172.5 µL345 µLV3 Pool 1 (10µM)2 µL55.2 µL110.4 µLNuclease-free water3 µL82.8 µL165.6 µLTotal11.5 µL310.5 µL621 µLandPool 2 setupComponentReaction 2 (each)Reaction 2 (3-col)Reaction 2 (6-col)Q5 Hot Start High-Fidelity 2X Master Mix6.25 µL172.5 µL345 µLV3 Pool 2 (10µM)2 µL55.2 µL110.4 µLNuclease-free water3 µL82.8 µL165.6 µLTotal11.5 µL310.5 µL621 µL
10
To prevent pre-PCR contamination the mastermix for each pool should be made up in the mastermix cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
ComponentReaction 1 (each)Reaction 1 (3-col)Reaction 1 (6-col)Q5 Hot Start High-Fidelity 2X Master Mix6.25 µL172.5 µL345 µLV3 Pool 1 (10µM)2 µL55.2 µL110.4 µLNuclease-free water3 µL82.8 µL165.6 µLTotal11.5 µL310.5 µL621 µL
ComponentReaction 2 (each)Reaction 2 (3-col)Reaction 2 (6-col)Q5 Hot Start High-Fidelity 2X Master Mix6.25 µL172.5 µL345 µLV3 Pool 2 (10µM)2 µL55.2 µL110.4 µLNuclease-free water3 µL82.8 µL165.6 µLTotal11.5 µL310.5 µL621 µL
?. [Post-PCR cleanup]
Spin down ARTIC PCR products in the Post-PCR lab.  Carefully remove seal.  Run the Bravo protocol to merge  of Reaction 1 and  of Reaction 2 into a single plate for SPRI cleanup.
7.5 µl
7.5 µl
?. [Post-PCR cleanup]
Perform a 1.0X bead cleanup protocol on Bravo with the following parameters per well magnetic beads 70% ethanol for two wash steps  Qiagen Buffer EB elution plate content Qiagen Buffer EB elution input Elution recoveryAfter SPRI, seal with PlateLoc and spin down before continuing.
15 µl
350 µl
15 µl
10 µl
9 µl
?. [Native barcoding]
ER/AT Master Mix calculations.  Make up in an Eppendorf tube, mix thorougly, then pipet to column 1 of the Post-PCR distribution plate.ComponentER/AT(each)ER/AT (3-col)ER/AT(6-col)Ultra II End Prep Buffer1.233.1266.24Ultra II End Prep Enzyme0.513.827.6Nuclease-free water5138276Total Volume6.7184.92369.84
ComponentER/AT(each)ER/AT (3-col)ER/AT(6-col)Ultra II End Prep Buffer1.233.1266.24Ultra II End Prep Enzyme0.513.827.6Nuclease-free water5138276Total Volume6.7184.92369.84
?. [Native barcoding]
Bravo will stamp  cleaned-up amplicons per ER/AT reaction.
3.3 µl
?. [Native barcoding]
For Native Barcoding, use the following quantities for 3-col or 6-col setup.  Mix thoroughly, spin down, then pipet to column 4 of the Post-PCR reagent plate for Bravo distribution.  Program will then add 0.75 uL ER/AT product and 1.25 barcode to each well.  Mix, seal, spin, and continue with protocol.ComponentLigation (each)Ligation (3-col)Ligation (6-col)Blunt/TA Ligase Master Mix5138276nuclease-free water382.8165.6Total Volume8220.8441.6
ComponentLigation (each)Ligation (3-col)Ligation (6-col)Blunt/TA Ligase Master Mix5138276nuclease-free water382.8165.6Total Volume8220.8441.6