TITLE: MiniXT protocol
AUTHORS: Marc-Aurel Fuchs, Arun Mahesh, Clara Radulescu, Deborah Lavin, Fiona Rogan, Julia Miskelly, Stephen Bridgett, David Simpson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We present the ‘mini-XT’ miniaturized tagmentation-based library preparation protocol used for Illumina WGS of SARS-CoV-2 positive samples. Reverse transcription and amplification is based upon the </span><a href="https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">nCoV-2019 sequencing protocol v3 (LoCost)V.3</span></a><span> by Josh Quick. </span></div><br/><div class = "text-block">The key new feature of the protocol is the use of acoustic liquid transfer to automate and reduce volumes during library preparation. It is optimized for the sequencing of 384 samples, offering reduced consumable use and costs and improved throughput.</div><br/></div>

[STEPS]
?. [cDNA synthesis]
ReagentFor 1 reaction (μL)For 420 reactions (μL, 4 x 96 well plate)Luna RT Supermix2840 Nuclease free water31260
ReagentFor 1 reaction (μL)For 420 reactions (μL, 4 x 96 well plate)Luna RT Supermix2840 Nuclease free water31260
?. [cDNA synthesis]
Thaw the RNA samples on ice.
?. [cDNA synthesis]
Shake the plates at 1200 RPM for 10 seconds, and spin at 280 xg for 1 min.
?. [cDNA synthesis]
Thaw LunaScript RT Supermix and nuclease free water on ice and at RT, respectively.
?. [cDNA synthesis]
Prepare the mastermix as show in Table 1 below:
?. [cDNA synthesis]
Incubate on a thermocycler using the conditions outlined in Table 2, ensuring that the lid is heated to 105 ºC:StepTemperature (ºC)Duration1252 min&nbsp;25520 min&nbsp;3951 min&nbsp;44∞
StepTemperature (ºC)Duration1252 min&nbsp;25520 min&nbsp;3951 min&nbsp;44∞
?. [cDNA amplification]
Prepare the Q5 PCR mastermixes (A and B, using Artic nCov2019 V3 primers) as outlined in Table 3 and keep on ice.
?. [cDNA amplification]
ReagentFor 1 reaction (μL)For 840 reactions (μL)Nuclease free water12.7510710Q5 reaction buffer54200dNTPs0.5420Primers (10 mM: A, or B)43360Q5 DNA polymerase0.25210
ReagentFor 1 reaction (μL)For 840 reactions (μL)Nuclease free water12.7510710Q5 reaction buffer54200dNTPs0.5420Primers (10 mM: A, or B)43360Q5 DNA polymerase0.25210
?. [Amplicon clean-up ]
Spin Pool A, Pool B 96-well plates at 280 xg, 1 min, RT.
?. [Amplicon clean-up ]
In a clean 96-well plate combine 10 μL of Pool A and 10 μL of Pool B. Pipette mix ten times.
?. [Amplicon quantification]
Spin the plate from Step 27 at 280 xg, 1 min, RT.
?. [Amplicon clean-up ]
Spin the A/B plate at 280 xg, 1 min, RT.
?. [Amplicon quantification]
Prepare the working solution from the Quant-iT™ dsDNA Assay Kit, broad range; for every 200 μL buffer add 1 μL dye. Vortex well to mix.
?. [Amplicon quantification]
Measure on a PHERAstar Omega plate reader: shake the plate for 30 sec before reading.
?. [Amplicon normalisation]
Prepare a .csv file containing:Echo plate position, Plate ID (A1, A2, B1, or B2), Well position on plate,Sample IDs, Quant-IT readings, and volume (in nL) to add to obtain amplicons at 0.2 ng/µL.
?. [Amplicon normalisation]
Change NTC transfer volumes to 500 nL.
?. [Amplicon normalisation]
Combine 30 μL from each well of the four 96-well plates into an Echo qualified flat-bottom 384-well polypropylene microplate 2.0 in the A1, A2, B1, B2 pooling style as shown in Table 5:
?. [Amplicon normalisation]
96-well plate (row)A1 (384 position)96-well plate (row)A2 (384 position)AA1 (A3, A5...)AA2BC1 (C3, C5...)BC2CE1 (E3, E5...)CE2DG1DG2EI1EI2FK1FK2GM1GM2HO1HO296-well plate (row)B1 (384 position)96-well plate (row)B2 (384 position)AB1AB2BD1BD2CF1CF2DH1DH2EJ1EJ2FL1FL2GN1GN2HP1HP2
96-well plate (row)A1 (384 position)96-well plate (row)A2 (384 position)AA1 (A3, A5...)AA2BC1 (C3, C5...)BC2CE1 (E3, E5...)CE2DG1DG2EI1EI2FK1FK2GM1GM2HO1HO296-well plate (row)B1 (384 position)96-well plate (row)B2 (384 position)AB1AB2BD1BD2CF1CF2DH1DH2EJ1EJ2FL1FL2GN1GN2HP1HP2
?. [Library preparation for Illumina sequencing]
Thaw Nextera XT DNA Library Preparation Kit reagents and decant into the following Echo source plates:
?. [Library preparation for Illumina sequencing]
Perform focus calibration and plate surveys on all Echo qualified flat-bottom 384-well polypropylene microplate 2.0 (as in Step 42).
?. [Library QC]
Label four 1.5 mL low-binding eppys:"Pool ID"“First clean”“Second clean”"Pool ID CLEAN”
?. [Illumina sequencing]
Sequencing is performed with a MiSeq Reagent Kit v2) (300 cycles PE):
?. [cDNA amplification]
For one RT plate, prepare one Pool A and one Pool B 96-well plate, where each well contains 22.5 μL of either A or B mastermix.
?. [cDNA amplification]
Incubate on a thermocycler using the conditions outlined in Table 4, ensuring that the lid is heated to 105 ºC:
?. [cDNA synthesis]
Add 5 μL mastermix to each well of a 95-well plate, followed by 5 μL RNA. Each RT plate must contain two no template controls (NTCs) in the form of 5 μL of nuclease free water. Pipette mix ten times upon addition.
?. [cDNA synthesis]
Spin the RT plate(s) at 1000 xg for 1 min.
?. [cDNA amplification]
StepTemperature (ºC)Duration198 30 sec29815 sec3635 minRepeat steps 1-3 34 times44∞º
StepTemperature (ºC)Duration198 30 sec29815 sec3635 minRepeat steps 1-3 34 times44∞
?. [cDNA amplification]
Add 2.5 μL of the RT product from Step 8, and pipette mix ten times.
?. [Amplicon clean-up ]
Transfer the supernatant to a clean 96-well plate, and store at 4 ºC.
?. [Amplicon clean-up ]
Keeping the plate on the magnet: perform two ethanol washes as follows:
?. [Amplicon clean-up ]
Remove and discard the supernatant, ensuring that you do not disturb the beads.
?. [Amplicon clean-up ]
Place the plate on a 96-well plate magnet for 3 min, RT.
?. [Amplicon clean-up ]
Incubate for 5 min, RT.
?. [Amplicon clean-up ]
Add 30 μL of KAPA Pure beads and pipette mix twenty times.
?. [Amplicon clean-up ]
Place the plate on a 96-well plate magnet for 3 min, RT.
?. [Amplicon clean-up ]
Incubate for 5 min, RT.
?. [Amplicon clean-up ]
Remove the plate form the magnet, and add 60 μL of 10 mM Tris pH 8.0 to each well. Pipette mix twenty times.
?. [Amplicon clean-up ]
After the second ethanol wash, air dry the beads for 4 min, RT.
?. [Amplicon clean-up ]
Add 140 μL of 80% ethanol to each well.
?. [Amplicon clean-up ]
Incubate for 30 sec, RT.
?. [Amplicon clean-up ]
Remove and discard the supernatant.
?. [Amplicon clean-up ]
After 1 min, remove any residual ethanol from the wells.
?. [Amplicon quantification]
Add 3 μL of clean amplicons to their respective wells.
?. [Amplicon quantification]
Vortex and spin down the standards, and add 3 μL to their respective wells.
?. [Amplicon quantification]
Prepare one plate for the standards (of which there are eight) by adding 100 μL of working solution in triplicate wells.
?. [Amplicon quantification]
For each plate from Step 27, prepare one black flat-bottom 96-well plate by adding 100 μL of working solution to each well.
?. [Amplicon quantification]
Read the standards first, generate the second polynomial fit, and save as a template to measure the sample plate(s).
?. [Amplicon quantification]
Quantify all NTCs using Qubit™ 1X dsDNA HS Assay Kit:
?. [Amplicon quantification]
Quantify all NTCs using TapeStation 4200 and Aligent D1000 screentape and reagents:
?. [Amplicon quantification]
Prepare the standards: 190 µL of mastermix + 10 µL Standard 1190 µL of mastermix + 10 µL Standard 2198 µL of mastermix + 2 µL Control.
?. [Amplicon quantification]
For each NTC:198 µL of mastermix + 2 µL NTC.
?. [Amplicon quantification]
Vortex and incubate for 2 min, RT, in the dark.
?. [Amplicon quantification]
Read on a Qubit 4 Fluorometer.
?. [Amplicon quantification]
To each 0.2 mL TapeStation 4200 eppy, add 3 µL sample buffer.
?. [Amplicon quantification]
Add 1 µL ladder or 1 µL NTC to the appropriate eppys.
?. [Amplicon quantification]
Vortex and spin down.
?. [Amplicon quantification]
Measure on TapeStation 4200 as per manufacturer's guidelines.
?. [Amplicon normalisation]
The maximum volume Echo can transfer is 10000 nL, therefore any samples with low Quant IT readings yielding volumes > 10000 nL must be changed to 10000 nL.
?. [Amplicon normalisation]
Perform a plate survey on the 384-well plate from Step 40, and perform focus calibration on the Echo.
?. [Amplicon normalisation]
For transfer exceptions, either: (a) create a rescue file, or (b) manually transfer to 0.2 ng/μL Echo qualified flat-bottom 384-well polypropylene microplate 2.0.
?. [Amplicon normalisation]
Label a new Echo qualified flat-bottom 384-well polypropylene microplate 2.0 as "0.2 ng/μL" and use the .csv file from Step 37 as your Echo "region definitions" to perform amplicon transfer.
?. [Amplicon normalisation]
To each well of 0.2 ng/μL Echo qualified flat-bottom 384-well polypropylene microplate 2.0, add 40 μL10 mM Tris pH 8.0 and pipette mix eight to ten times.
?. [Amplicon normalisation]
Spin the plate from Step 45 at 280 xg, 1 min, RT, and store at 4 ºC.
?. [Illumina sequencing]
Prepare the pool for the MiSeq as outlined below:
?. [Library preparation for Illumina sequencing]
2 mL TD buffer into one well of an Echo 525 liquid handler source plate.
?. [Library preparation for Illumina sequencing]
63 μL of ATM into A1, B1, to H1 (inclusive) of an Echo qualified flat-bottom 384-well polypropylene microplate 2.0.
?. [Library preparation for Illumina sequencing]
Full volume of NT buffer into one well of an Echo 525 liquid handler source plate.
?. [Library preparation for Illumina sequencing]
Full volume of NPM buffer into one well of an Echo 525 liquid handler source plate.
?. [Library preparation for Illumina sequencing]
Using the Echo, perform the following sequential transfers:
?. [Library preparation for Illumina sequencing]
Spin the plate from Step 49 at 280 xg, 1 min, RT, and store at 4 ºC.
?. [Library preparation for Illumina sequencing]
1 μL of TD buffer to each well of an Eppendorf Twin tec PCR plate, 384 wells.
?. [Library preparation for Illumina sequencing]
500 nL of amplicons (From Step 46) to corresponding wells of an Eppendorf Twin tec PCR plate, 384 wells.
?. [Library preparation for Illumina sequencing]
500 nL of ATM to each well of an Eppendorf Twin tec PCR plate, 384 wells.
?. [Library preparation for Illumina sequencing]
For transfer exceptions, see Step 44.
?. [Library preparation for Illumina sequencing]
Incubate on a thermocycler for 5 min, at 55 ºC.
?. [Library preparation for Illumina sequencing]
Incubate for 5 min, RT.
?. [Library preparation for Illumina sequencing]
Transfer 500 nL of NT buffer to each well of an Eppendorf Twin tec PCR plate, 384 wells.
?. [Library preparation for Illumina sequencing]
Transfer 1.5 μL of NPM to each well of an Eppendorf Twin tec PCR plate, 384 wells.
?. [Library preparation for Illumina sequencing]
Transfer 1 μL of pre-mixed forward and reverse indexes (or 500 nL of each seperately) to corresponding wells of an Eppendorf Twin tec PCR plate, 384 wells.
?. [Library preparation for Illumina sequencing]
Incubate on a thermocycler using the conditions outlined in Table 6, ensuring that the lid is heated to 105 ºC:
?. [Library preparation for Illumina sequencing]
StepTemperature (ºC)Duration1723 min29530 sec39510 sec45530 sec57230 sec6725 min74∞
StepTemperature (ºC)Duration1723 min29530 sec39510 sec45530 sec57230 sec6725 min74∞
?. [Library QC]
Place the eppy on a magnetic stand for 2-5 min until the supernatant is clear.
?. [Library QC]
Incubate for 10 min, RT.
?. [Library QC]
Add 80 μL KAPA Pure beads and pipette mix twenty to thirty times.
?. [Library QC]
Transfer 50 μL to "First clean" eppy.
?. [Library QC]
Pool each well from this row into the "Pool ID" eppy, and pipette mix ten times.
?. [Library QC]
Pool 2.5 μL from each well of the plate from Step 50 into one row of a 96-well plate.
?. [Library QC]
Repeat Steps 55 to 63.
?. [Library QC]
Transfer elute to "Second clean" eppy.
?. [Library QC]
Place the eppy on a magnetic stand for 2-5 min until the supernatant is clear.
?. [Library QC]
Incubate for 10 min, RT.
?. [Library QC]
Add 50 µL 10 mM Tris pH 8.0 and pipette mix ten times.
?. [Library QC]
Air dry beads for 8 min, RT.
?. [Library QC]
Keeping the eppy on the magnetic stand, wash beads twice with 80% EtOH. For each wash:Add 400 µL to the eppy, Incubate for 30 sec to 1 min, RT, Remove and discard the supernatant
?. [Library QC]
Remove and discard the supernatant.
?. [Library QC]
Quantify the cleaned pool using TapeStation 4200 and Aligent D1000 screentape and reagents as in Step 36.
?. [Library QC]
Quantify the cleaned pool (in duplicate) using Qubit™ 1X dsDNA HS Assay Kit as in Step 35.
?. [Library QC]
Transfer elute to "Pool ID, CLEAN" eppy.
?. [Illumina sequencing]
Thaw the contents of Box 1 of 2 1h before use.
?. [Illumina sequencing]
Store Box 2 of 2 at 4 ºC until use.
?. [Illumina sequencing]
Calculate the molarity of the pool, and the amount of pool and 10 mM Tris pH 8.5 (in µL) required to dilute to 4 nM, using:(a) the Qubit measurements for the Pool (from Step 67),(b) the average fragment size (bp) from the TapeStation (From Step 68), and the following formula: =(1000000*(1/average fragment size)*(1/660))*AVERAGE(Qubit measurments)
?. [Illumina sequencing]
Prepare the MiSeq sample sheet as follows:
?. [Illumina sequencing]
Add Pool ID to "Experiment Name", and "Sample Project" column.
?. [Illumina sequencing]
Add Sample IDs from Step 37. to "Description" column.
?. [Illumina sequencing]
Add sample barcodes to "Sample ID" column.
?. [Illumina sequencing]
Add the plate ID to "Sample Plate" column.
?. [Illumina sequencing]
Prepare the 4 nM pool using the values obtained in Step 71. Vortex and spin down.
?. [Illumina sequencing]
Prepare 0.2 N NaOH.
?. [Illumina sequencing]
Incubate for 5 min, RT.
?. [Illumina sequencing]
In an eppy labelled "20 pM" combine 5 µL of 4 nM pool and 5 µL0.2 N NaOH.
?. [Illumina sequencing]
Add 990 µL HT1 buffer to 20 pM eppy. Vortex and spin down.
?. [Illumina sequencing]
In an eppy labelled "9 pM" combine 330 µL HT1 buffer and 270 µL from the 20 pM eppy. Vortex and spin down.
?. [Illumina sequencing]
In an eppy labelled "Load" combine 594 µL from the 9 pM eppy with 6 µLf 12.5 pM PhiX (control template). Vortex and spin down.
?. [Illumina sequencing]
Prepare the flow cell, incorporation buffer as per manufacturer's guidelines, and prepare the MiSeq as instructed on the screen.
?. [Illumina sequencing]
Add 600 µL from Step 72.7 to the reagent cartridge as per manufacturer's guidelines.
?. [Bioinformatics]
Perform Basecalling and Demultiplexing of the sequencing data using the Illumina "MiSeq Reporter software" (or alternatively the Illumina "bcl2fastq" or "BCL Convert").
?. [Bioinformatics]
Use the "connor-lab/ncov2019-artic-nf" nextflow-based open-source pipeline, as used by the COG-UK consortium, (from: https://github.com/connor-lab/ncov2019-artic-nf) to perform the QC, read-trimming, alignment to Wuhan reference, and generate consensus fasta sequence. Use the '--profile conda' option with the '--illumina' option
?. [Bioinformatics]
Use the Pangolin open-source software (from: https://github.com/cov-lineages/pangolin ) to call the Lineage for each sample's consensus fasta sequence.