TITLE: UCSF CAT COVID-19 Tailed 275bp v3 ARTIC protocol v1
AUTHORS: Delsy   Martinez, Tyler   Miyasaki, Eric Chow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>This is a tailed amplicon method similar to </span><a href="https://www.protocols.io/view/sars-cov-2-tailed-amplicon-illumina-sequencing-bipikdke" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/sars-cov-2-tailed-amplicon-illumina-sequencing-bipikdke</span></a><span> but based on the shorter 275nt amplicons in </span><a href="https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bh4zj8x6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bh4zj8x6</span></a><span>. One modification from the Gohl trailed protocol is the use of partial TruSeq tails instead of Nextera tails and using two vs four multiplexed pools. The advantage of this method is the speed and low cost due to several reasons. </span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Only two separate cDNA ARTIC PCR reactions are required.</li><li style = "counter-reset:ol0;">After the indexing PCR step using TruSeq indexing primers, equal volumes are pooled and a single tube bead cleanup is performed. </li><li style = "counter-reset:ol0;">These libraries only require PE150 sequencing.</li></ol></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The workflow can be completed in under 8 hour. If a small number of samples are prepared, a MiSeq Nano or MIcro run can be used, for a total time from RNA to FASTQs in roughly 24 hours. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Note that this primer design has a 15bp primer overlap region around 20.5kb that results in 15 bases that cannot be sequenced with this method. We are investigating other primers in this region to recover these bases.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">We have optimized this method to process samples in 384-well format using a Beckman Biomek i7 and a Labcyte Echo to minimize tip usage (three tips per sample). The procedure can also be performed manually at lower scale or in 96-well format, but you may need to increase the volume of the reactions for more accurate pipeting. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">A cost effective manner to purchase this oligo set is with IDT oPools. At list price, you can order the two pools for a roughly $200 each. This is enough to prepare 500 samples at our volumes. You can also list each oligo two in a pool to get twice the amount of oligo at twice the cost.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [cDNA generation]
Important! This step must be performed in a RNase free, pre-PCR environment in which post PCR COVID-19 amplicons are not present, to minimise risk of sample contamination.Decontaminate bench surfaces, pipettes and gloves with RNase ZAP before starting work. Keep reagents and samples chilled throughout the process.
?. [cDNA generation]
Defrost at least 10ul RNA. Store at 4C
?. [cDNA generation]
Add 8ul RNA to 2ul  and mix by pipetting.
?. [cDNA generation]
Seal and briefly centrifuge plate.
?. [cDNA generation]
Place plate on a thermocycler and run the following program:  AB1TemperatureTime2  25°C  2 minutes3  55°C  20 minutes  4  95°C  1 minute  5  4°C   ∞  6  Lid temp: Tracking       If using a Labcyte Echo, we perform the RT reaction in an LDV source plate with a flat block thermocycler and transfer cDNA directly into the ARTIC multiplex PCR reactions in the next step.
AB1TemperatureTime2  25°C  2 minutes3  55°C  20 minutes  4  95°C  1 minute  5  4°C   ∞  6  Lid temp: Tracking
?. [275bp ARTIC cDNA amplification]
Note: Primers for this protocol were modified from https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bh4zj8x6. Note that amplicons 109 and 100 have a shared primer region that results in a 15bp overlap that will be unrecoverable. Partial TruSeq R1 or R2 were added to the forward and reverse primers in the pool. Primer sequences and additional information can be found here:   These primers can be ordered individually or as an IDT oPool using the the linked form. You can get 50pmol/oligo (one copy per oligo) or 100pmol/oligo (two copies per oligo) if you double each line in the file. These will be enough for 500 or 1000 samples using 10ul PCR reactions. For larger numbers of samples, it will be more cost effective to order each oligo separately. Create a 10uM pool for primer set 1 and primer set 2.
?. [275bp ARTIC cDNA amplification]
Add 1ul of cDNA template to each PCR plate. Seal the plates, vortex to mix, and spin down.
?. [275bp ARTIC cDNA amplification]
Note: Amplification should ideally be performed in a different lab to minimize the risk of contaminating other samples.     As noted in the original protocol (https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bh4zj8x6), you may need to adjust the annealing/extension temperature on your own thermocycler.   ABC1StepTemperatureTime21  98°C    30 seconds  32  98°C    15 seconds  4363°C  5 minutes  54Repeat steps 2 &amp; 3 for a total of 35 cycles65  4°C    ∞  If desired, you can check your amplified cDNA by electrophoresis. You should expect products around 300 bp. For high Ct samples, we observe more primer dimers.
ABC1StepTemperatureTime21  98°C    30 seconds  32  98°C    15 seconds  4363°C  5 minutes  54Repeat steps 2 &amp; 3 for a total of 35 cycles65  4°C    ∞
?. [275bp ARTIC cDNA amplification]
PAUSE POINT Amplified cDNA can be stored at 4°C (overnight) or -20°C (up to a week).
?. [Perform indexing PCR]
Prepare indexing PCR reaction in 384-well formatIndexing PCR MixVol/PCR RXN (µl)NEB Ultra II Q5 mix55uM TruSeq i7/i5 Indexing primer mix2Water2Total9
Indexing PCR MixVol/PCR RXN (µl)NEB Ultra II Q5 mix55uM TruSeq i7/i5 Indexing primer mix2Water2Total9
?. [Pool samples and cleanup]
Pool 0.5ul of each library. We transfer the PCR reactions into an Echo LDV plate and use the Echo to pool. If transferring manually, you can increase the volume per sample, just make sure to scale the Ampure bead cleanup appropriately.
We pool samples after PCR and perform a single cleanup on the pool. If desired, each sample can be purified separately, quantified, and then pooled.
?. [Pool samples and cleanup]
Add a 0.8x volume of  or similar reagent. Mix by pipeting. Allow the DNA to bind for 5 minutes.
?. [Perform indexing PCR]
Combine the two cDNA PCR reactions from each sample and dilute 1:100 in water.
?. [Perform indexing PCR]
Add 1ul of the diluted cDNA pool to the indexing PCR mix.
?. [Perform indexing PCR]
Seal plate, vortex to mix, spin down.
?. [Perform indexing PCR]
Thermocycle according to the protocol below. StepTemperatureTime1  98°C  0:302  98°C  0:103  65°C  1:154Repeat Step 2&3 for 10 cycles total5  65°C  5:006  4°C   ∞
StepTemperatureTime1  98°C  0:302  98°C  0:103  65°C  1:154Repeat Step 2&3 for 10 cycles total5  65°C  5:006  4°C   ∞
?. [275bp ARTIC cDNA amplification]
Prepare the two multiplexed SARS-CoV-2 master mixes:  AB1Multiplexed master mixVol/PCR RXN (µl)2NEB Q5 Hot Start High-Fidelity mix*53Pool 1 or Pool 2 primer mix (10uM)1.64Nuclease-free water  2.45Total9Aliquot 9ul of Pool 1 and Pool 2 mix into two separate 384-well PCR plates.*NEB Ultra II Q5 mix may also be used instead, but temperatures may need to be adjusted.
AB1Multiplexed master mixVol/PCR RXN (µl)2NEB Q5 Hot Start High-Fidelity mix*53Pool 1 or Pool 2 primer mix (10uM)1.64Nuclease-free water  2.45Total9
?. [Pool samples and cleanup]
Place the tube on a magnet.After the beads have bound to the side of the tube, remove the supernatant.
?. [Pool samples and cleanup]
While keeping the tube on the magnet, add 70% ethanol without disturbing the beads.Wait 30 seconds and remove the ethanol.
?. [Pool samples and cleanup]
Perform a second ethanol washWhile keeping the tube on the magnet, add 70% ethanol without disturbing the beads.Wait 30 seconds and remove the ethanol.
?. [Pool samples and cleanup]
Wait 5 minutes or until beads are dry.Resuspend the beads in one volume water or elution buffer.Wait 30 secondsPlace tube on magnet. After beads have moved to the side of the tube, transfer eluate to a new tube.
?. [QC samples and sequence]
Quantify your samples using a method of your choice (such as Qubit) and run your samples on an electrophoresis system such as a BioAnalyzer or TapeStation (you may need to dilute your sample). You should expect to see your library centered around 400bp.Samples should be run on an appropriate PE150 system depending on how many total reads are required. For lower Ct samples, 100,000 reads is sufficient. For higher Ct samples, 1,000,000 or more reads may be required. We have sequenced 384 samples on a single NovaSeq SP PE150 lane or 768 on a single NovaSeq S1 lane. For analysis, make sure you trim reads using the 275bp primer panel sequences instead of the standard 400bp ARTIC panel sequences.If you have many high Ct samples, primer dimers may be more abundant and a second bead cleanup could be required.