TITLE: Targeted Sequencing by Sanger to Recover Key Mutations in Illumina SARS-CoV-2 Whole-genome Sequences
AUTHORS: Lavanya Singh, Pius M. Brzoska, Ugochukwu    J. Anyaneji, James Emmanuel San, Tulio De Oliveira

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Spike (S) gene of SARS-CoV-2 demonstrates more frequent mutations in an otherwise conserved genome.  With the proliferation of SARS-CoV-2 variants, there is an urgent need to continuously monitor the novel mutations arising to investigate viral diversity and vaccine escape mutants.  Next-generation sequencing (NGS) technologiesthat are predominantly used to generate whole-genome sequences due to cost-effectiveness are prone to insufficient coverage and gaps in the sequence products. Sanger sequencing technology presents a complementary solution to enhance NGS sequencing through quick, gene-specific sequencing that has the potential to mitigate or avoid some of these challenges. Here we present sequencing primers that we designed and an associated workflow to recover key mutations (K417N, E484K, N501Y) in the receptor-binding domain of the 501Y.V2 lineage recently identified in South Africa.</div><div class = "text-block"><span style = "font-weight:bold;">The Sanger sequencing CE workflow</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dhuzbeh6f.jpg" /></div></div>

[STEPS]
?. [Abstract]
In a 96-well plate, add 10 μl of master mix to each sample and no-template control (NTC) well.
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One-step RT-PCR
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Add 10 μl of extracted RNA to each sample well.  Mix gently by pipetting
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?. On ice, prepare a master mix containing the components from Table 1 (add 10% extra for pipetting overage).
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On ice, prepare a master mix containing the components from Table 1 (add 10% extra for pipetting overage).Table 1 – One-step RT-PCR Master Mix ComponentVol x1 (µl)TaqPath 1-step RT-qPCR master mix (ThermoFisher Scientific)5501Y.V2 forward primer (5uM)*1501Y.V2 reverse primer (5uM)*1Nuclease-free water3Total10* Primer details are shown in Table 2Table 2 – PCR and sequencing 501Y.V2 primer details (accession: EPI_ISL_678597)ForwardGATCTCTGCTTTACTAATGTCTATGCAGATReverseGCTGGTGCATGTAGAAGTTCAAAAGAmplicon Start22698Amplicon End23098Length401 base pairs
Table 1 – One-step RT-PCR Master Mix ComponentVol x1 (µl)TaqPath 1-step RT-qPCR master mix (ThermoFisher Scientific)5501Y.V2 forward primer (5uM)*1501Y.V2 reverse primer (5uM)*1Nuclease-free water3Total10
Table 2 – PCR and sequencing 501Y.V2 primer details (accession: EPI_ISL_678597)ForwardGATCTCTGCTTTACTAATGTCTATGCAGATReverseGCTGGTGCATGTAGAAGTTCAAAAGAmplicon Start22698Amplicon End23098Length401 base pairs
?. [Sanger Sequencing]
In a 96-well plate, add 10 μl of master mix to each sample and no-template control (NTC) well.
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Add 10 μl of extracted RNA to each sample well.  Mix gently by pipetting
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Add 10 μl nuclease-free water to the NTC well.  Mix gently by pipetting
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Seal the 96-well plate and briefly centrifuge the plate to collect the contents at the bottom of the wells.
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Place the 96-well plate on a thermocycler and run the PCR condition from Table 3.Table 3 – One-step Thermocycling ConditionsStepTemperature (°C)Time CyclesUNG incubation252 minutes1RT incubation5015 minutes1Enzyme activation952 minutes1Amplification953 seconds406030 secondsHold4
Table 3 – One-step Thermocycling ConditionsStepTemperature (°C)Time CyclesUNG incubation252 minutes1RT incubation5015 minutes1Enzyme activation952 minutes1Amplification953 seconds406030 secondsHold4
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PCR Purification
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In a new 96-well plate, add 10 µl of the PCR product to 4 µl Exo-SAP IT express reagent on ice.
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Mix by gentle pipetting, seal the plate and briefly centrifuge to collect the contents at the bottom of the wells.
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Run the following thermocycling conditions (Table 4).Table 4 - Exo-SAP Express IT Thermocycling conditionsStepTemperature (°C)Time Digest374 minutesHeat deactivation801 minutesHold4hold
Table 4 - Exo-SAP Express IT Thermocycling conditionsStepTemperature (°C)Time Digest374 minutesHeat deactivation801 minutesHold4hold
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Cycle sequencing
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On ice, prepare a master mix according to the components in Table 5, for each forward and reverse primer.Table 5 - BigDye™ Terminator cycle sequencing reactionComponentVolume (μl)BigDye™ Terminator 3.1 Ready Reaction Mix2BigDye™ Terminator v1.1 &amp; v3.1 5X Sequencing Buffer1501Y.V2 forward OR reverse (3.2uM)1Nuclease-free water 5Total volume9ul
Table 5 - BigDye™ Terminator cycle sequencing reactionComponentVolume (μl)BigDye™ Terminator 3.1 Ready Reaction Mix2BigDye™ Terminator v1.1 &amp; v3.1 5X Sequencing Buffer1501Y.V2 forward OR reverse (3.2uM)1Nuclease-free water 5Total volume9ul
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Add 9 µl of cycle sequencing master mix to each sample / NTC well of a 96-well plate.
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Add 1 µl of purified PCR product to each well.
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Mix gently by pipetting.
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Prepare an internal positive pGEM control by mixing the components from Table 6 and adding to a designated well of the 96-well plate.Table 6 – pGEM internal control reaction volumeComponentVolume (μl)BigDye™ Terminator 3.1 Ready Reaction Mix4M13 (-21) Primer2pGEM Control DNA1Deionized water3Total volume10
Table 6 – pGEM internal control reaction volumeComponentVolume (μl)BigDye™ Terminator 3.1 Ready Reaction Mix4M13 (-21) Primer2pGEM Control DNA1Deionized water3Total volume10
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Seal the plate, briefly centrifuge and place the plate in a thermocycler to run the conditions from Table 7.Table 7 - Cycle sequencing thermocycling conditions	PCR stepTemperature (°C)TimeCyclesInitial denaturation961 minute1Denaturation9610 seconds25Annealing505 secondsExtension604 minutesHold4∞1
Table 7 - Cycle sequencing thermocycling conditions	PCR stepTemperature (°C)TimeCyclesInitial denaturation961 minute1Denaturation9610 seconds25Annealing505 secondsExtension604 minutesHold4∞1
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Cycle sequencing reaction purification
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Using the BigDye Xterminator Purification kit, prepare a master mix according to the volumes in Table 8.  Prepare an additional 10% for pipetting overage.Table 8 – BigDye XTerminator MastermixComponentVol x1 (µl)SAM solution45XTerminator Solution10Total55
Table 8 – BigDye XTerminator MastermixComponentVol x1 (µl)SAM solution45XTerminator Solution10Total55
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Ensure that the solution is properly mixed and add 55 µl to each well.
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Seal the plate and vortex at 1800 rpm for 30 minutes.
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Centrifuge the plate at 1000 xg for 2 minutes.
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Proceed with capillary electrophoresis on a DNA sequencer.