TITLE: ARTIC-NEB: SARS-CoV-2 Library Prep
AUTHORS: Lusajo Mwakibete, Cristina Tato, Vida Ahyong, Amy Kistler, Manu Vanaerschot, Angela Detweiler, Michelle Tan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This experiment was carried out to investigate the effect of fragmentation time on the genome recovery of SARS-COV2 from COVID-19 positive patient samples using an iSeq. The experiment uses the ARTIC protocol to attain the amplicon from the extracted RNA, followed by FS DNA Library Prep Kit to make the amplicon library. We investigated the fragmentation time at the beginning of the FS DNA library prep. Fragmentation time of 5 minutes gave the best genome coverage having the least dropout regions. </div><div class = "text-block">The ARTIC protocol was adapted from Josh Quick 2020.nCoV-2019 sequencing </div><div class = "text-block"><a href="https://www.protocols.io/view/ncov-2019-sequencing-protocol-v2-bdp7i5rn/dx.doi.org/10.17504/protocols.io.bdp7i5rn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocol.io</span></a></div></div>

[STEPS]
?. [Dilution of Input Viral RNA]
Diluting samples based on Ct values to prevent overloading.Caution! Carryout the following dilution steps with DNAse/RNAse free water in PCR-pre, RNAse free environment (Biosafety cabinet). RNA was extracted using the Zymo Quick DNA/RNA Viral MagBead Kit (R2141). RT-PCR CT values dictate the viral RNA input as following:  AB11-151:10216-35no dilutionDISCLAIMERAll reactions are 0.5X of the manufacturer's protocol. Half reactions can be performed and still yield genomes whilst saving on reagents.
AB11-151:10216-35no dilution
?. [cDNA ]
Random Hexamer PrimingSet up the following reaction (8-Strip Tube).   AB10.5 rxn vol stock (µL)260µM Random hexamers (NEB S1330S)0.5310 mM dNTPS (NEB N0447)0.54RNA Input Sample5.55Total Volume6.56µL/rxnMix the mixture with a pipette thoroughly, place the reaction in the thermocycler with the following setting:  ABC1TemperatureTime2Step 165°C5 min3Step 24°CHold4Lid105°C
AB10.5 rxn vol stock (µL)260µM Random hexamers (NEB S1330S)0.5310 mM dNTPS (NEB N0447)0.54RNA Input Sample5.55Total Volume6.56µL/rxn
ABC1TemperatureTime2Step 165°C5 min3Step 24°CHold4Lid105°C
?. [cDNA ]
Reverse Transciptase (RT)Make the following master mix and add to the Random Primed RNA  AB10.5 rxn vol stock (µL)2SuperScript IV 5X Buffer23100mM DTT0.54RNase OUT RNAse Inhibitor0.55Superscript IV Reverse Transcriptase0.56Total Volume3.57µL/rxnMix the mixture with a pipette thoroughly, place the reaction in the thermocycler with the following setting:  ABC1TemperatureTime2Step 125°C5min3Step 242°C50min4Step 370°C10min5Step 44°Chold6Lid105°C◊ checkpoint: can freeze overnight (-20°C)◊
AB10.5 rxn vol stock (µL)2SuperScript IV 5X Buffer23100mM DTT0.54RNase OUT RNAse Inhibitor0.55Superscript IV Reverse Transcriptase0.56Total Volume3.57µL/rxn
ABC1TemperatureTime2Step 125°C5min3Step 242°C50min4Step 370°C10min5Step 44°Chold6Lid105°C
?. [cDNA Amplicon Amplification]
MULTIPLEX PCR The ARTIC primer list (V3) can be found here - https://github.com/artic-network/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019.tsv. Primers can be purchased as pool from IDT reps as well. Lyophilized primers were resuspended to a concentration of 100 uM.Primers were pooled in "Pool 1" or "Pool 2" according to ARTIC protocol 1. 100 uM primer pools were further diluted to 10uM. 10µM primers can be pre-aliquoted and stored at -20°C. The table below was used to prepare separate master mixes for Pool 1 and Pool 2 primers respectively.  AB10.5 rxn vol stock (µL)2NEBNext Ultra II Q5 Hot Start 2x master mix6.253Primer pool at 10 µM (1 or 2)24Nuclease-free water35cDNA from Step 31.256Total Volume12.57µL/rxnMix the mixture with a pipette thoroughly, place the reaction in the thermocycler with the following setting:  ABCD1TemperatureTimeCycle2Step 198°C30 sec3Step 298°C15 sec30*463°C5 min5Step 34°CHold6Lid105°C*Number of cycles depends on the input RNA Ct values. 30 cycles works for all Ct values, lower sample Ct values less cycles required.
AB10.5 rxn vol stock (µL)2NEBNext Ultra II Q5 Hot Start 2x master mix6.253Primer pool at 10 µM (1 or 2)24Nuclease-free water35cDNA from Step 31.256Total Volume12.57µL/rxn
ABCD1TemperatureTimeCycle2Step 198°C30 sec3Step 298°C15 sec30*463°C5 min5Step 34°CHold6Lid105°C
?. [cDNA Amplicon Amplification]
Pool and SPRI (Magnetic Bead Purification) CleanPool multiplexed samples from pool 1 and pool 2 together for each respective sample. *allow beads to sit in RT for 30 mins priorCombine Pool 1 and Pool 2 PCR reactions together from the prior multiplex PCR step. Total volume will be 25µL.  Use SPRI bead  1x ratio of beads-to-total volume of sample. Prep 80% EtOH.Add 25µL of room temperature beads (1x) to the pooled PCR reaction (25µL). Mix well.Pulse spin the tubes, but be sure not to spin down beads. Incubate for 5 mins at room temperature.Place samples on magnetic rack, and incubate for 5 mins on the rack.Remove supernatant.Add 200µL of 80% EtOH to samples while on the magnetic rack. Incubate at room temperature for 1 min then remove the supernatant.Repeat EtOH wash step for total of 2 times.Air dry the beads for 5 mins while on the magnetic rack.Remove tube from magnetic rack. Elute DNA from beads into 69µL of 0.1x TE Buffer, 10mM Tris-HCl, or Nuclease free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place on magnetic rack until solution is clear ~ 5 mins.Remove 67µL of the supernatant and transfer to a clean nuclease free PCR tube.◊ checkpoint: can freeze overnight (-20°C)◊
?. [cDNA Amplicon Amplification]
Quantification of AmpliconQuantify amplicon using a fragment analyzer such as TapeStation and BioAnalyzer. Use Qubit dsDNA High Sensitivity (HS) assay to get amplicon concentration prior to loading sample into fragment analyzer, to make sure you don't overload the ScreenTape/Chip. The concentration quantification aids with selecting DNA input for the NEBNext Ultra II FS DNA Library Prep which follows. A peak should be spotted at 400bp which coincides to the size of the amplified amplicons of the SARS-COV-2 genome.
?. [Amplicon Library Prep]
Using FS DNA Library Kit (E7805,E6177) to finish amplicon libraries. Make sure the input Amplicon concentration is between 10 -100ng. FragmentationMake the following master mix (The reaction has been scaled down to 0.5X from the normal protocol FS DNA Library Prep Kit.  AB10.5X rxn vol stock (µL)2DNA input (10-100ng)133NEBNext Ultra II FS Reaction Buffer (Yellow)3.54NEBNext Ultra II FS Enzyme Mix (Yellow)15Total Volume17.5Mix the mixture with a pipette thoroughly, place the reaction in the thermocycler with the following setting:  ABC1TemperatureTime2Step 1 (Fragmentation)37°C5 min3Step 2 (Enzyme Deactivation)65°C30 min4Step 34°CHold5Lid75°C
AB10.5X rxn vol stock (µL)2DNA input (10-100ng)133NEBNext Ultra II FS Reaction Buffer (Yellow)3.54NEBNext Ultra II FS Enzyme Mix (Yellow)15Total Volume17.5
ABC1TemperatureTime2Step 1 (Fragmentation)37°C5 min3Step 2 (Enzyme Deactivation)65°C30 min4Step 34°CHold5Lid75°C
?. [Amplicon Library Prep]
Adaptor Ligation Add the following components directly to the prior FS reaction mixture.   AB10.5X rxn vol stock (µL)2FS Reaction Mixture17.53NEBNext Ultra II Ligation Master Mix* (Red)154NEBNext Ligation Enhancer (Red)0.55NEBNext Adaptor for Illumina (1:100 dilution)1.256Total Volume34.25*add adaptor separately from the master mix to avoid adapter dimers. Mix the mixture with a pipette thoroughly, place the reaction in the thermocycler with the following setting:  ABC1TemperatureTime2Step 120°C15 min3Step 24°CHold4LidOff
AB10.5X rxn vol stock (µL)2FS Reaction Mixture17.53NEBNext Ultra II Ligation Master Mix* (Red)154NEBNext Ligation Enhancer (Red)0.55NEBNext Adaptor for Illumina (1:100 dilution)1.256Total Volume34.25
ABC1TemperatureTime2Step 120°C15 min3Step 24°CHold4LidOff
?. [Amplicon Library Prep]
SPRI (Magnetic Bead Purification) Clean*allow beads to sit in RT for 30 mins priorUse SPRI bead  0.9x ratio of beads-to-total volume of sample. Prep 80% EtOH.Add 30.83µL of room temperature beads (0.9x) to Adaptor Ligation reaction (34.25µL). Mix well.Pulse spin the tubes, but be sure not to spin down beads. Incubate for 5 mins at room temperature.Place samples on magnetic rack, and incubate for 5 mins on the rack.Remove supernatant.Add 200µL of 80% EtOH to samples while on the magnetic rack. Incubate at room temperature for 1 min then remove the supernatant.Repeat EtOH wash step for total of 2 times.Air dry the beads for 5 mins while on the magnetic rack.Remove tube from magnetic rack. Elute DNA from beads into 9µL of 0.1x TE Buffer, 10mM Tris-HCl, or Nuclease free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place on magnetic rack until solution is clear ~ 5 mins.Remove 7µL of the supernatant and transfer to a clean nuclease free PCR tube.◊ checkpoint: can freeze overnight (-20°C)◊
?. [Amplicon Library Prep]
PCR ENRICHMENT/BARCODING  AB10.5X rxn vol stock (µL)2Purified, adaptor-ligated cDNA63USER Enzyme (Cat no. M5505L, 250uL)1.54NEBNext Ultra II Q5 Master Mix (Blue)12.55i7 barcoded primer (NEB index primer/TruSeq/or similar)*2.56i5 barcoded primer (NEB Universal primer/TruSeq/or similar)*2.57Total volume25*The i7 and i5 primers can be premixed, each primer should be 5µM in the Primer MixMix the mixture with a pipette thoroughly, place the reaction in the thermocycler with the following setting:  ABCD1TemperatureTimeCycle2Step 137°C15 min13Step 298°C30 sec14Step 398°C30 sec12565°C75 sec6Step 465°C5 min17Step 54°CHold* depending on the RNA input number of cycles can be varied accordingly. (I.E. High cDNA amplicon input, less cycles and vice-versa).
AB10.5X rxn vol stock (µL)2Purified, adaptor-ligated cDNA63USER Enzyme (Cat no. M5505L, 250uL)1.54NEBNext Ultra II Q5 Master Mix (Blue)12.55i7 barcoded primer (NEB index primer/TruSeq/or similar)*2.56i5 barcoded primer (NEB Universal primer/TruSeq/or similar)*2.57Total volume25
ABCD1TemperatureTimeCycle2Step 137°C15 min13Step 298°C30 sec14Step 398°C30 sec12565°C75 sec6Step 465°C5 min17Step 54°CHold
?. [Amplicon Library Prep]
SPRI (Magnetic Bead Purification) Clean*allow beads to sit in RT for 30 mins priorUse SPRI bead  0.8x ratio of beads-to-total volume of sample. Prep 80% EtOH.Add 20µL of room temperature beads (0.8x) to the PCR enriched/barcoded library reaction (25µL). Mix well.Pulse spin the tubes, but be sure not to spin down beads. Incubate for 5 mins at room temperature.Place samples on magnetic rack, and incubate for 5 mins on the rack.Remove supernatant.Add 200µL of 80% EtOH to samples while on the magnetic rack. Incubate at room temperature for 1 min then remove the supernatant.Repeat EtOH wash step for total of 2 times.Air dry the beads for 5 mins while on the magnetic rack.Remove tube from magnetic rack. Elute DNA from beads into 27µL of 0.1x TE Buffer, 10mM Tris-HCl, or Nuclease free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place on magnetic rack until solution is clear ~ 5 mins.Remove 25µL of the supernatant and transfer to a clean nuclease free PCR tube.◊ checkpoint: can freeze overnight (-20°C)◊
?. [Quality Control of Amplicon Library]
Quantification of Amplicon LibraryQuantify library using a fragment analyzer such as TapeStation and BioAnalyzer. Use DNA HS Qubit to get library concentration prior to loading sample into fragment analyzer, to make sure you don't overload the machine. The profile of the library will aid in showing if there are any contaminants in your library such as adapter dimers at around 140bp. In addition, more SPRI clean-ups might be required if adapter dimers are present in the library. The clean amplicon library should look as following.
?. [Analysis ]
Analysis of Fragmentation Time :Insert Size: The fragmentation time varied the insert sizes for our libraries. 5 mins and 10 mins fragmentation times decreased a majority of our insert size from around 400bp to 200-250bp. This is good for individuals who want to sequence the SARS-CoV-2 genome with an ISEQ whilst using the artic protocol. This will allow the 2 by 150 reads, to attain better reads spanning the entire genome, with the shorter fragment sizes. Thus we suggest the fragmentation time of 5 mins instead of 10 minutes because we don't want to overfragment our amplicon inserts. Depth Coverage: