TITLE: nCoV-2019 environmental sample sequencing protocol
AUTHORS: Sam Diaz-Munoz, Ivy Jose, AJ Campbell

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ARTIC amplicon MinION sequencing protocol for nCoV-2019 starting from environmental samples</div><div class = "text-block">This protocol is a modification of the nCoV-2019 sequencing protocol v2 (GunIt) V.2, a one-pot native barcoding protocol was developed by Josh Quick in conjunction with Oxford Nanopore Technologies, New England Biolabs and BCCDC. </div><div class = "text-block">This version modifies a few steps from the above protocol to obtain a better yield from low concentration samples.</div><div class = "text-block">This protocol was implemented in the manuscript: "Environmental Surveillance for SARS-CoV-2 at the UC Davis Medical Center" by David Coil et al. Code for data analysis here:</div><div class = "text-block"><a href="https://github.com/sociovirology/sars_cov2_environmental_seq" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/sociovirology/sars_cov2_environmental_seq</span></a></div><div class = "text-block">This protocol was developed by the Díaz-Muñoz Lab: Sam Díaz-Muñoz, Ivy José, and A.J. Campbell.</div></div>

[STEPS]
?. [cDNA preparation]
Mix the following components in an 0.2mL 8-strip tube;Component                                        Volume50µM random hexamers                 10mM dNTPs mix (10mM each)    Template RNA                                   Total
1 µl
1 µl
11 µl
13 µl
Viral RNA input from a clinical sample should be between Ct 18-35. If Ct is between 12-15, then dilute the sample 100-fold in water, if between 15-18 then dilute 10-fold in water. This will reduce the likelihood of PCR-inhibition.
A mastermix should be made up in the mastermix cabinet and aliquoted into PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
?. [cDNA preparation]
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
?. [cDNA preparation]
Incubate the reaction as follows: for Place on ice for
65 °C
?. [cDNA preparation]
Add the following to the annealed template RNA: Component	                                Volume SSIV Buffer	                                 100mM DTT                                  RNaseOUT RNase Inhibitor 	 SSIV Reverse Transcriptase	 Total
4 µl
1 µl
1 µl
1 µl
20 µl
A mastermix should be made up in the mastermix cabinet and added to the denatured RNA in the extraction and sample addition cabinet. Tubes should be wiped down when entering and leaving the mastermix cabinet.
?. [cDNA preparation]
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
?. [cDNA preparation]
Incubate the reaction as follows:Hold at
42 °C
70 °C
5 °C
?. [Primer pool preparation]
If required resuspend lyophilised primers at a concentration of 100µM each. The ARTIC nCoV-2019 V3 Panel can be ordered pre-mixed from IDT.
nCov-2019/V3 primers for this protocol were designed using Primal Scheme and generate overlapping 400nt amplicons. Primer names and dilutions are listed in the table below.
?. [Primer pool preparation]
Generate primer pool stocks by adding  of each odd region primer to a  Eppendorf labelled “Pool 1 (100µM)” and each even region primer to a  Eppendorf labelled  “Pool 2 (100µM)”. The pool is also given in the .TSV files in the primalscheme output. These are your 100µM stocks of each primer pool.
5 µl
1.5 mL
1.5 mL
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Primer pool preparation]
Dilute this primer pool 1:10 in molecular grade water, to generate 10µM primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination.
Primers need to be used at a final concentration of 0.015µM per primer. In this case V3 pools have 110 primers in pool 1 and 108 primers in pool 2. so the requirement is ~4µL primer pool (10uM) per 25µL reaction. For other schemes, adjust the volume added appropriately.
?. [Multiplex PCR]
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8-strip PCR tubes:Component                                             Pool 1                Pool 2 5X Q5 Reaction Buffer                           10 mM dNTPs                                         Q5 Hot Start DNA Polymerase             V3 Primer Pool 1 or 2 (10µM)               Nuclease-free water                              Total
5 µl
5 µl
0.5 µl
0.5 µl
0.25 µl
0.25 µl
4 µl
4 µl
12.75 µl
12.75 µl
22.5 µl
22.5 µl
A PCR mastermix for each pool should be made up in the mastermix cabinet and aliquoted into PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
?. [Multiplex PCR]
In the extraction and sample addition cabinet add  cDNA to each tube and mix well by pipetting.
2.5 µl
The extraction and sample addition cabinet should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Multiplex PCR]
Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
?. [Multiplex PCR]
Set-up the following program on the thermal cycler:  Step                        Temperature     Time                    CyclesHeat Activation         1Denaturation              25-35 Annealing                   25-35Hold                                   Indefinite              1
98 °C
98 °C
65 °C
4 °C
Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35
?. [Pool PCRs & Concentrate Products]
Label new PCR strip tubes by sample. For guidance on low concentration samples, see 16.1.Add 20ul of each Pool 1 PCR reaction into the new tubes, being careful to ensure sample identity. Add 20ul of each Pool 2 PCR reaction into the new tubes, being careful to ensure sample identity. 40ul total volume.ermine and record the concentrations of the PCR products: Qubit, nanodrop, etc.
?. [Quantification and normalisation]
Label another  Eppendorf tube for each sample.
1.5 mL
Input to the one-pot native barcoding reaction is 50ng per sample. Process at least 6 samples plus one negative control per library in order to have sufficient material to load on the sequencer at the end.
?. [Native barocoding]
Barcode the amplicon pools using the one-pot native barcoding approach. *For guidance on low concentration samples, see 20.1, labeled with an '*"
{"blocks":[{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"9r429","text":"This one-pot native barcoding protocol was developed in conjunction with Oxford Nanopore Technologies, New England Biolabs and BCCDC.","type":"unstyled"}],"entityMap":[]}
?. [Native barocoding]
Quantify  of the barcoded amplicon pool using the Quantus Fluorometer using the ONE dsDNA assay.
1 µl
?. [Native barocoding]
Set up the following AMII adapter ligation and clean-up with SFB.
{"blocks":[{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"qnlk","text":"This is a subprotocol for performing adapter ligation with AMII","type":"unstyled"}],"entityMap":[]}
?. [Native barocoding]
Quantify  of the final library using the Quantus Fluorometer using the ONE dsDNA assay.
1 µl
Final library can be now be stored in 10 mM Tris pH 8 at 4°C for up to a week if needed otherwise proceed directly to MinION sequencing.
?. [MinION sequencing]
Prime the flowcell and load  sequencing library onto the flowcell.
20 ng
From experience we know 20 ng is optimum loading input for short amplicons.
?. [MinION sequencing]
Start the sequencing run using MinKNOW.
?. [Multiplex PCR]
Determine and record the concentrations of the PCR products: Qubit, nanodrop, etc.
?. [Pool PCRs & Concentrate Products]
Follow the directions for size selection and purification using the Zymo Research Select a Size DNA Concentrator Kit. * Use the ≥ 100 bp cutoff. Elute in   Elution buffer and incubate for  at room temperature before final spin.
10 µl
?. [Pool PCRs & Concentrate Products]
Determine and record the concentrations of the cleaned and concentrated PCR products: Qubit, nanodrop, etc.
?. [Multiplex PCR]
Optional: If the post-PCR concentrations are low, run a second PCR reaction with both pool 1 and pool 2 primers on each sample, as above, and combine the products. This should yield more DNA going into the clean and concentrate step.
?. [Pool PCRs & Concentrate Products]
If low PCR product concentrations are a concern, the complete volume of the  Pool 1 and Pool 2 reactions may be combined to proceed with into the clean and concentrate steps. If a second PCR reaction for each sample has been performed, the complete volume of these may be pooled to yield a total volume of ~ 100ul per sample going into clean and concentrate reaction.
?. [Native barocoding]
Optional: For low concentration samples, a second barcoding reaction may be performed for each sample, using the End Prepped sample as template. In the protocol referenced in step 20, 'One-pot barcoding of amplicons v2', this is step 3 (or step 20.3 of this protocol). *Be careful adding the barcodes and template to ensure sample identity is retained!* Combine the 2 barcoding reactions to yield a total volume of ~ 40ul per barcoded sample.