TITLE: High-Throughput Tiled Amplicon Sequencing of Sars-CoV2 using Seqwell&#39;s plexWell 384
AUTHORS: Megan Folkerts, mnguyen , Danielle Vazquez,  Amber Jones

[STEPS]
?. [cDNA synthesis]
For cDNA synthesis of viral RNA,  follow steps 1-6 of the v2  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 samples prior to cDNA conversion, and noticed no issues with PCR inhibition by not doing so. Samples were diluted prior to GS pcr. See below.
?. [GS amplification with ARTIC primers]
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 diluted in step 2, follow steps 6-9 of the ARTIC protocol above to complete gene-specific PCR. 35 cycles should be used for PCR
?. [cDNA synthesis]
Because the plexWell protocol requires precise sample inputs, cDNA must be diluted prior to gene-specific PCR according to the following:  AB1N2 CTDilution212-151:1000315-181:100418-281:10528+no dilution Dilute in 10mM Tris HCl, 0.05% Tween 20, or 10mM Tris HCL.
AB1N2 CTDilution212-151:1000315-181:100418-281:10528+no dilution
?. [GS amplification with ARTIC primers]
Perform one 1X and one 0.7X Ampure XP bead cleanups on the combined PCR product according to the following:
Because plexWell requires precise DNA input amounts, cleanups must be performed to ensure dimer removal for accurate quantitation. Do not simply dilute GS product as in the ARTIC protocol.
?. [GS amplification with ARTIC primers]
Following GS pcr, combine full volume of reaction 1 with full volume of reaction 2 (final volume, 50ul)
?. [GS amplification with ARTIC primers]
Following bead-cleanup, perform a Qubit spot-check of the concentrations of post-GS product across the plate. Typically, quantifying 15-20 samples of a 96-well plate is sufficent to calculate the global dilution factor for the subsequent step.Alternatively, if high-throughput  quantification methods are available (such as a fluorescent plate reader) quantify the entire plate using a fluorescent assay of choice (such as pico green).
?. [GS amplification with ARTIC primers]
Average DNA input of samples going into the plexWell protocol should be 2.5ng/ul. If quantification at step 22 reveals the average concentration of samples is greater than 2.5ng/ul, perform the following plate-wide global dilution:Global dilution factor = Average sample concentration/2.5ngPerform dilution of all samples in plate by the above factor using 10mM Tris HCL. Avoid EDTA-containing solutions when diluting.
The above is taken from Seqwell's protocol note on use of the plexWell 384 kit with Sars-CoV2 sequencing and the ARTIC protocol. https://seqwell.com/wp-content/uploads/2020/05/20200526_plexWell_ARTIC_protocol_20200527.pdfThe DNA inputs for each plate going into the plexWell system must be very precise to ensure correct fragment sizes are obtained at the end of the protocol. Adding too much DNA to the initial tagmentation step can cause fragment sizes to be too large for efficient sequencing on certain short-read Illumina systems.
?. [plexWell384 initial tagmentation and i7 addition]
Before beginning the plexWell protocol, bring the following reagents to room temperature:  ABC1ReagentStorage
  conditionsFormat2SBP/X96 Plate-20CPlate/Red3MAGwise Paramagnetic Beads4CTube4Pool Barcode Reagent-20CTube/Red5Library Primer Mix-20CTube/Natural
ABC1ReagentStorage
  conditionsFormat2SBP/X96 Plate-20CPlate/Red3MAGwise Paramagnetic Beads4CTube4Pool Barcode Reagent-20CTube/Red5Library Primer Mix-20CTube/Natural
?. [GS amplification with ARTIC primers]
Pour beads into reservoir for use with a 96 well plate.
?. [GS amplification with ARTIC primers]
For each reaction, pipette 50 uL beads into the reaction wells.
?. [GS amplification with ARTIC primers]
Mix by slowly pipetting up and down at least 10 times. Make sure solution is completely homogenous.
?. [GS amplification with ARTIC primers]
Let the mixture sit on the bench for 5 minutes for the beads to bind the DNA.
?. [GS amplification with ARTIC primers]
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.
?. [GS amplification with ARTIC primers]
Discard the liquid from wells without touching the beads.
?. [GS amplification with ARTIC primers]
Dispense 200µl 80% ethanol into each well without disturbing beads while the plate is sitting on a magnetic plate.
?. [GS amplification with ARTIC primers]
Incubate for 30 seconds. If you disturbed the beads, then let the mixture stand for 2 minutes.
?. [GS amplification with ARTIC primers]
Discard the ethanol without touching the beads.
?. [GS amplification with ARTIC primers]
Dispense another 200µl 80% ethanol into each well, without disturbing beads, while the plate is sitting on a magnetic plate.
?. [GS amplification with ARTIC primers]
Incubate for 30 seconds. If you disturbed the beads, then let the mixture stand for 2 minutes.
?. [GS amplification with ARTIC primers]
Discard the ethanol without touching the beads.  Make sure all ethanol is discarded as it can interfere with DNA elution.
?. [GS amplification with ARTIC primers]
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).
?. [GS amplification with ARTIC primers]
Pipette 32.5 uL of pre-warmed 10mM Tris HCL, 0.05% Tween 20 onto dry wells and pipette beads up and down to bring them into solution.
?. [GS amplification with ARTIC primers]
Place beads on a magnetic plate and let beads collect for 3 minutes.
?. [GS amplification with ARTIC primers]
Remove 30ul (leaving behind the 2.5uL extra volume) of eluted DNA into a new PCR reaction plate without touching the beads. The beads have no more DNA bound to them.
?. [plexWell384 initial tagmentation and i7 addition]
Add 4ul of gene-specific product (diluted if necessary according to step 23 guidelines) to the appropriate wells of an SBP/X96 plateSlowly pipette to mix. Try not to introduce excess bubbles.
Steps 25-67 detail the protocol for Seqwell's plexWell 384 kit. The full user guide can be found here:https://seqwell.com/wp-content/uploads/2020/02/plexWell_384_and_96_Library_Preparation_Kit_User_Guide.pdfProtocol deviations from the above pdf will be noted in the steps below.
?. [plexWell384 initial tagmentation and i7 addition]
Add 4ul of Coding Buffer into the wells of the SBP/X96 plateSlowly pipette to mix. Try not to introduce excess bubbles. Seal plate and centrifuge at 1000XG for 1 minute.
At this point, depending on the model of thermal cycler being used, you may need to transfer the contents of the reaction to a mid-skirt plate.
?. [plexWell384 initial tagmentation and i7 addition]
Run the following program on a thermal cycler, with heated lid set to 100C  AB1TemperatureTime255C15 min310Chold
AB1TemperatureTime255C15 min310Chold
?. [plexWell384 initial tagmentation and i7 addition]
Centrifuge plate at 1000G for 1 minute to remove condensation from seal. Dispense 6ul of solution X into each well of the plate.Slowly pipette to mix. Try not to introduce excess bubbles. Seal plate and centrifuge at 1000XG for 1 minute.
?. [plexWell384 initial tagmentation and i7 addition]
Run the following program on a thermal cycler, with heated lid set to 100C:  AB1TemperatureTime268C10min&nbsp;325Chold
AB1TemperatureTime268C10min&nbsp;325Chold
?. [plexWell384 initial tagmentation and i7 addition]
Centrifuge plate at 1000G for 1 minute to remove condensation from seal. Combine 9ul of each reaction into a 2ml LoBind tube.
?. [plexWell 384 pool purification]
Vortex MAGwise Paramagnetic Beads thoroughly to ensure beads are resuspended in solution.
?. [plexWell 384 pool purification]
Add 850 uL (or ~approx. 1 volume equivalent) of MAGwise to the tube with the pooled SB reaction.
?. [plexWell 384 pool purification]
Pipette mix solution and incubate on benchtop for 5 minutes
?. [plexWell 384 pool purification]
Place tube on magnetic stand and wait for bead pellet to form (~3 minuntes)
?. [plexWell 384 pool purification]
Remove and discard supernatant. Do not disturb the pellet.
?. [plexWell 384 pool purification]
With the beads on the magnet, add two volumes of ethanol. (~1.7mL, or as much as is needed to cover the beads) Incubate for 30 seconds
?. [plexWell 384 pool purification]
Remove and discard supernatant
Complete this step quickly. Do not allow beads to dry out during wash process
?. [plexWell 384 pool purification]
Repeat steps 37-38 for a total of 2 washes
?. [plexWell 384 pool purification]
Cap the tube of beads, vortex, and spin briefly to bring residual ethanol to bottom of tube
?. [plexWell 384 pool purification]
Immediately place on magnet for beads to settle (~30 seconds)
?. [plexWell 384 pool purification]
Pipette out any residual ethanol
?. [plexWell 384 pool purification]
Add 40 uL of 10 mM Tris-HCl to bead pellet, remove from magnetic, pipette mix, and incubate on bench top for 5 min
?. [plexWell 384 pool purification]
Return tube to magnetic stand and wait for bead pellet to form (~2 minutes)
?. [plexWell 384 pool purification]
Carefully transfer out 39 uL of DNA eluate to a PCR tube [NOTE: This is your PRODUCT]
?. [plexWell 384 pool purification]
Quantify your pool with pico green or Qubit HS reagents.
Pool should quantify between 4-8ng/ul. If you are outside this range, dilute pool to achieve desired concentration. Correct concentration is essential to obtain products of appropriate size for sequencing.
?. [plexWell384 Pool barcoding/i5 addition]
Add 9ul PB reagent to PCR tube with the purified SB reaction pool. Take note of which pool barcode was used. Pipette thoroughly to mix.
The addition of 9ul PB reagent differs from the Seqwell protocol for the plexWell kit, and is necessary to achieve fragment sizes compatible with short-read sequencing.
This protocol is designed for the high-throughput sequencing of Sars-CoV-2 samples. For multiplexing purposes, ensure that a different pool barcode is used for each plate of 96 (up to 12 uniquely barcoded plates/pools total). Seqwell currently provides 1152 unique index combinations for this purpose.
?. [plexWell384 Pool barcoding/i5 addition]
Add 22ul Coding Buffer to the tube and pipette thoroughly to mix. Cap the tube, vortex, and centrifuge briefly.
?. [plexWell384 Pool barcoding/i5 addition]
Run the following program on a thermal cycler, with heated lid set to 100C  AB1TemperatureTime255C15min325Chold
AB1TemperatureTime255C15min325Chold
?. [plexWell384 Pool barcoding/i5 addition]
Briefly centrifuge tube. Add 33ul of X solution to the reaction. Pipette slowly to mix.Re-cap tube and centrifuge briefly
?. [plexWell384 Pool barcoding/i5 addition]
Run the following program on a thermal cycler, with heated lid set to 100C  AB1TemperatureTime268C10 min325Chold
AB1TemperatureTime268C10 min325Chold
?. [plexWell 384 PB pool purification]
Vortex and spin PB reaction, and transfer contents to a 1.5ml LoBind tubeVortex Magwsie beads thoroughly to ensure beads are resuspended in solution
?. [plexWell 384 PB pool purification]
Add 99ul MAGwise beads to the tube with the pooled SB reaction
?. [plexWell 384 PB pool purification]
Follow steps 33-41 to clean the SB reaction
?. [plexWell 384 PB pool purification]
Add 24ul of 10mM Tris-HCL to bead pellet, remove from magnetic stand, pipette to mix, and incubate on benchtop for 5 minutes
?. [plexWell 384 PB pool purification]
Return tube to magnetic stand and wait for bead pellet to form (~2 minutes)
?. [plexWell 384 PB pool purification]
Carefully transfer 23ul of DNA eluate to a new PCR tube.
The transfer of some beads with the purified product with not inhibit library amplification
?. [plexWell 384 Library amplification]
Add 4ul of Library Primer Mix to the purified product
?. [plexWell 384 Library amplification]
Add 27ul of Kapa HiFi Hotstart ReadyMix and mix well by pipetting.Close PCR tube and centrifuge briefly.
?. [plexWell 384 Library amplification]
Run the following program on a thermal cycler with heated lid set to 100C  ABC1TemperatureTimeNotes272C10
  min395C3
  min498C30
  secRepeat steps 4-6 for 12 cycles total564C15
  sec672C30
  sec772C3
  min84Chold
ABC1TemperatureTimeNotes272C10
  min395C3
  min498C30
  secRepeat steps 4-6 for 12 cycles total564C15
  sec672C30
  sec772C3
  min84Chold
?. [plexWell384 Library purification]
Briefly centrifuge library amplification reaction and measure total volume using a pipette. Transfer product to LoBind tube.
?. [plexWell384 Library purification]
Dilute the library to a final volume of 205ul with 10mM Tris-HCL pH 8.0 and pipette to mix
?. [plexWell384 Library purification]
Transfer 100ul of diluted library to a new 1.5ml LoBind tube for clean-up. Store the remaining 105ul at -20C until QC is complete.
?. [plexWell384 Library purification]
Vortex MAGwise beads thoroughly.Add 75 ul (or ~.75 volumes) of MAGwise beads to the 100 ul of diluted amplified library
This volume differs from the Seqwell protocol for the plexWell kit, and is necessary to achieve fragment sizes compatible with short-read sequencing
?. [plexWell384 Library purification]
Follow steps 33-41 to purify library amplification product
?. [plexWell384 Library purification]
Add 32ul 10mM Tris-HCL to bead pellet, remove from magnet, pipette to mix, and incubate on bench top for 5 minutes
?. [plexWell384 Library purification]
Return to magnetic stand and wait for bead pellet to form (~2 minutes)
?. [plexWell384 Library purification]
Carefully transfer 28ul of DNA eluate to a new 1.5ml LoBind tube. This is the final purified library.
?. [Library QC]
Visualize final library on a tapestation or bioanalyzer using high-sensitivity reagents.
?. [Library QC]
Quantify final library using Kapa Quant reagents and an appropriate real-time PCR machine. Quantify multiple dilutions of each final library to ensure accurate readings.Alternatively, qubit final library in triplicate to determine concentration.
?. [Sequencing]
Pool 12 (up to 1152 samples total) uniquely barcoded pools from steps 1-69 above at equimolar concentrations
?. [Sequencing]
Quantify final pool of pools using Kapa Quant reagents and an appropriate real-time PCR machine. Quantify multiple dilutions of each final library to ensure accurate readings.Alternatively, qubit final library in triplicate to determine concentration.
?. [Sequencing]
Visualize final pool of pools on a bioanalyzer or fragment analyzer to determine final pool size. Pool may need to be diluted prior to running on HS kit. Alternatively, if expected concentration is high enough, DNA 1000 reagents may be used.
?. [Sequencing]
Sequence pool of pools (1152 samples total) on a NextSeq High-Output kit, using 150 X 150 bp paired end reads, and 8bp dual index reads. Added PhiX should be 5% of the total run.Detailed loading instructions for Illumina NextSeq500/550 can be found here: https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/nextseq/nextseq-denature-dilute-libraries-guide-15048776-09.pdf
We typically target a cluster density of 160-200k/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 ~350bp in size, a loading concentration of 1.5 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.
?. [GS amplification with ARTIC primers]
Repeat steps 5-22, using 35ul beads in step 7 for a second, 0.7X cleanup.