TITLE: Rapid Tiled Amplicon Sequencing of Sars-CoV-2 using Illumina&#39;s DNA Prep
AUTHORS: Megan Folkerts, mnguyen , Danielle Vazquez,  Amber Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol for rapid, low-throughput (24 samples or fewer) sequencing of Sars-CoV-2-containing samples using Illumina's DNA prep, and the ARTIC protocol for tiled amplicon sequencing. Turnaround time for this protocol from raw sample to sequence data is ~35 hours, making it ideal for use when rapid elucidation of samples is necessary. </div></div>

[STEPS]
?. [cDNA synthesis]
For cDNA synthesis,  follow steps 1-6 of the v2 of nCoV-2019 sequencing protocol by Josh Quick dx.doi.org/10.17504/protocols.io.bdp7i5rn .
A new version of this protocol has been released which replaces ThermoFisher's SSIV reverse transcriptase with NEB's Lunascript. Though this provides significant cost savings, in our limited trial, the SSIV RT significantly outperformed the Lunascript.
We did not dilute based on CT value prior to conversion, and noted no issues with inhibition.
?. [ARTIC Gene-specific PCR]
GS amplification with ARTIC primers is based on version 2 of the nCoV-2019 protocol by Josh Quick (dx.doi.org/10.17504/protocols.io.bdp7i5rn) with modifications. Using cDNA obtained in step 1, follow steps 6-9 of the ARTIC protocol above to complete gene-specific PCR. 35 cycles should be used for PCR
?. [ARTIC Gene-specific PCR]
Following gene-specific PCR, for each sample, combine full volume of reaction 1 with full volume of reaction 2, for a total of 50ul.
?. [ARTIC Gene-specific PCR]
Perform 1 1X Ampure XP bead cleanup as follows:
?. [ARTIC Gene-specific PCR]
Pour beads into reservoir for use with a 96 well plate.
?. [ARTIC Gene-specific PCR]
For each reaction, pipette 50 uL beads into the reaction wells.
?. [ARTIC Gene-specific PCR]
Mix by slowly pipetting up and down at least 10 times. Make sure solution is completely homogenous.
?. [ARTIC Gene-specific PCR]
Let the mixture sit on the bench for 5 minutes for the beads to bind the DNA.
?. [ARTIC Gene-specific PCR]
Place the plate on a magnetic plate and let it stand for 5 minutes for the beads to collect on the bottom of the plate.
?. [ARTIC Gene-specific PCR]
Discard the liquid from wells without touching the beads.
?. [ARTIC Gene-specific PCR]
Dispense 200µl 80% ethanol into each well without disturbing beads while the plate is sitting on a magnetic plate.
?. [ARTIC Gene-specific PCR]
Incubate for 30 seconds.  If you disturbed the beads, then let the mixture stand for 2 minutes.
?. [ARTIC Gene-specific PCR]
Discard the Ethanol without touching the beads.
?. [ARTIC Gene-specific PCR]
Dispense another 200µl 80% ethanol into each well, without disturbing beads, while the plate is sitting on a magnetic plate.
?. [ARTIC Gene-specific PCR]
Incubate for 30 seconds.  If you disturbed the beads, then let the mixture stand for 2 minutes.
?. [ARTIC Gene-specific PCR]
Discard the ethanol without touching the beads. Make sure all ethanol is discarded as it can interfere with DNA elution.
?. [ARTIC Gene-specific PCR]
Place the plate on the bench and air dry beads for ~5 minutes or until beads appear dry (over-dried beads appear as a cracked, dried surface).
?. [ARTIC Gene-specific PCR]
Pipette 32.5 uL of pre-warmed 10mM Tris HCL, with 0.05% Tween 20 onto dry wells and pipette beads up and down to bring them into solution.
?. [ARTIC Gene-specific PCR]
Place beads on a magnetic plate and let beads collect for 3 minutes.
?. [ARTIC Gene-specific PCR]
Remove 30ul (leaving behind the 2.5uL extra) of eluted DNA into a new PCR reaction plate without touching the beads. The beads have no more DNA bound to them.
?. [QC of PCR product]
Qubit each sample and determine concentration post-cleanup.
?. [QC of PCR product]
In a new plate or 8-strip tube, dilute each sample as necessary with MBG H20 so that total input amount going into Illumina DNA Prep is 100-500ng, and final sample volume is 30ul.
For high-CT samples, it may not be possible to input 100ng. If this is the case, aim for the highest input possible without using all of gene-specific product (in the event that troubleshooting is necessary).
?. [Illumina DNA prep- tagmentation]
Steps 23-58 detail the protocol for Illumina's DNA Prep. The full protocol can be found here: https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/illumina_prep/illumina-dna-prep-reference-guide-1000000025416-09.pdfPrior to beginning Illumina DNA prep, bring the following reagents to room temperature. EPM should be thawed, but remain on ice throughout protocol.  AB1ReagentStorage&nbsp;2BLT2C-8C3TB1&nbsp;-25C4Index Adapter Plate&nbsp;-25C5Sample Purification Beads2C-8C6RSB-25C
AB1ReagentStorage&nbsp;2BLT2C-8C3TB1&nbsp;-25C4Index Adapter Plate&nbsp;-25C5Sample Purification Beads2C-8C6RSB-25C
?. [Illumina DNA prep- tagmentation]
Vortex BLT thoroughly, and combine the following reagents to prepare the tagmentation master mix. Reagent overage is included.  AB1ReagentVolume2BLT&nbsp;113TB111
AB1ReagentVolume2BLT&nbsp;113TB111
?. [Illumina DNA prep- tagmentation]
Vortex the mastermix, and add 20ul master mix to the 30ul diluted DNA from step 22. Pipette to mix. Cap tubes and seal plate, but do not centrifuge. Bead mixture needs to remain suspended.
?. [Illumina DNA prep- tagmentation]
Run the following program on a thermal cycler, with heated lid set to 100C.  AB1TemperatureTime255C15 minutes310Chold
AB1TemperatureTime255C15 minutes310Chold
?. [Illumina DNA prep- tagmentation]
Remove plate from thermal cycler. Add 10ul TSB to each reaction. Slowly pipette to resuspend beads. Seal plate. Do not centrifuge.
?. [Illumina DNA prep- tagmentation]
Run the following program on a thermal cycler, with heated lid set to 100C.  AB1TemperatureTime237C10 minutes310Chold
AB1TemperatureTime237C10 minutes310Chold
?. [Illumina DNA prep- tagmentation clean-up]
Remove seal, and place the plate on a magnetic stand. Wait until liquid is clear (~1-3 minutes)
?. [Illumina DNA prep- tagmentation clean-up]
Using a multichannel pipette, remove supernatant and discard.
?. [Illumina DNA prep- tagmentation clean-up]
Remove plate from magnet. Add 100ul TWB, pipetting very slowly to resuspend beads to avoid foaming.
?. [Illumina DNA prep- tagmentation clean-up]
Place the plate back on the magnetic stand, and wait until liquid clears (1-3 minutes).
?. [Illumina DNA prep- tagmentation clean-up]
Using a multichannel pipette, remove supernatant and discard.
?. [Illumina DNA prep- tagmentation clean-up]
Repeat steps 31-33 for a total of 2 washes.
?. [Illumina DNA prep- tagmentation clean-up]
Add 100ul of TWB to beads. Pipette slowly to resuspend beads to avoid foaming. Place plate back on magnetic stand and proceed to step 36.
?. [Illumina DNA prep- Library amplification]
While plate is on magnet, make the following master mix. Reagent overage is included in volumes listed.  AB1ReagentVolume2EPM223Nuclease-free water22
AB1ReagentVolume2EPM223Nuclease-free water22
?. [Illumina DNA prep- Library amplification]
Vortex briefly, and centrifuge mastermix for 10 secounds on a table-top centrifuge.
?. [Illumina DNA prep- Library amplification]
Remove supernatant from plate while still on the magnetic stand.
Some foam may remain, and will not affect library quality.
?. [Illumina DNA prep- Library amplification]
Add 40ul mastermix from step 36 to the beads. Pipette to mix until beads are completely resuspended.
?. [Illumina DNA prep- Library amplification]
Pierce the foil of the adapter index plate with a P200 pipette tip. Add 10ul of pre-prepaired i7 and i5 index adapters from the appropriate wells of the 96 plex plate to the bead mix. Be sure to record which indices are used.
?. [Illumina DNA prep- Library amplification]
Mix thoroughly by pipetting, seal plate, and centrifuge at 280g for 30 seconds.
?. [Illumina DNA prep- Library amplification]
Place on thermal cycler, and run the following program with heated lid set to 100C  ABC1TemperatureTimeCycles268C3
  minutes398C3
  minutes498C45
  secondsRepeat step 4-6 for a total of 5-12 cycles. Cycle number selection guide is in note below.562C30
  seconds668C2
  minutes768C1
  minute810Chold
ABC1TemperatureTimeCycles268C3
  minutes398C3
  minutes498C45
  secondsRepeat step 4-6 for a total of 5-12 cycles. Cycle number selection guide is in note below.562C30
  seconds668C2
  minutes768C1
  minute810Chold
PCR cycle number is dependent on original DNA input. Select PCR cycle number based on the following:  AB1DNA
  input (ng)Cycle number21 to 912310 to 248425 to 496550 to 5005
?. [Illumina DNA Prep- Library purification]
Centrifuge plate at 280g for 1 minute
?. [Illumina DNA Prep- Library purification]
Place the plate on a magnet and wait for the liquid to clear (5 minutes)
?. [Illumina DNA Prep- Library purification]
Transfer 45 ul of supernatant to a new PCR plate
?. [Illumina DNA Prep- Library purification]
Vortex SPB thoroughly to resuspend
?. [Illumina DNA Prep- Library purification]
Add 81ul of SPB to each well of the plate containing 45ul supernatant. Pipette to mix. Incubate on bench top for 5 minutes to allow beads to bind DNA.
?. [Illumina DNA Prep- Library purification]
Place plate on magnet and wait until the liquid clears (5 minutes)
?. [Illumina DNA Prep- Library purification]
Without disrupting the beads, remove the supernatant and discard.
?. [Illumina DNA Prep- Library purification]
With the plate still on the magnet, add 200u of fresh (
?. [Illumina DNA Prep- Library purification]
Incubate for 30 seconds
?. [Illumina DNA Prep- Library purification]
Remove and discard ethanol supernatant.
?. [Illumina DNA Prep- Library purification]
Repeat steps 50-51 for a total of 2 washes.
?. [Illumina DNA Prep- Library purification]
Use a p20 pipette to remove all residual ethanol. Air-dry plate on the magnet stand for 5 minutes, or until beads appear dry, but not cracked.
?. [Illumina DNA Prep- Library purification]
Remove plate from magnetic stand, and add 32ul room-temperature RSB to the beads. Pipette to resuspend.
?. [Illumina DNA Prep- Library purification]
Incubate at room temperature for 2 minutes.
?. [Illumina DNA Prep- Library purification]
Place the plate on the magnetic stand and wait for liquid to clear (2 minutes).
?. [Illumina DNA Prep- Library purification]
Transfer 30ul of supernatant to a new 96-well PCR plate. These are your final libraries.
?. [Library QC and pooling.]
If DNA input to Illumina DNA prep was 100ng or greater for ALL libraries, pool equal volumes of each library (up to 24 total) in a new 1.5 ml microcentrifuge tube. This is your final pool. If DNA input of ANY sample was less than 100ng, Qubit all samples.Based on qubit values, combine all samples (up to 24 total) equimolarly in a new 1.5ml microcentrifuge tube. This is your final pool.
?. [Library QC and pooling.]
Visualize final pool on a bioanalyzer or tape station to determine final library size.
You may need to dilute library prior to running high-sensitivity assays. Typically, 1:5 is sufficient to acheive results. Alternatively, use a DNA 1000 kit.
?. [Sequencing]
Sequence final pool on a MiSeq v2 nano kit, with 250 X 250 bp paired-end reads, and dual 8bp indexing. Added phiX should be 5% of the total run. Up to 24 total samples can be sequenced on a single MiSeq nano 500 cycle kit. Sequencing greater than 24 samples will result in insufficient viral genome coverage per sample.Detailed loading instructions for Illumina MiSeq can be found here:  https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/miseq/miseq-denature-dilute-libraries-guide-15039740-10.pdf
We typically target a cluster density of 600-800k/mm2. This is slightly less than what Illumina recommends. We've found that having a higher Q30 but less overall data due to lower clustering is more desirable than having a lower Q30 but more overall data, particularly for amplicon sequencing. You can adjust this based on your own preferences.
We found that, for a pool of ~300bp in average size, a loading concentration of 5 pM produced optimal results. Loading concentrations can vary slightly from machine to machine, however, and are dependent on overall library fragment size. You should adjust your loading concentration accordingly.