TITLE: nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon)
AUTHORS: Nikki Freed, Olin Silander

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To enable faster, easier sequencing of SARS-COV2 genomes with fewer steps than current methods, we use multiplexed 1200 base pair PCR amplicons with the Oxford Nanopore RAPID barcoding kit (RBK004). </div><div class = "text-block">This is a modification of the ARTIC amplicon V3 sequencing protocol for MinION for nCoV-2019 developed by Josh Quick, which produces 400 base pair amplicons and uses the Oxford Nanopore Ligation barcoding kit (LSK-109). </div><div class = "text-block">We have increased the size of the amplicons to 1200bp and use the RAPID barcode kit (RBK004), which enables requires less time and fewer reagents than the LSK-109 protocol. The amplicons produced in this protocol could also be used for Illumina sequencing. </div><div class = "text-block"><span>Primers were all designed using Primal Scheme: </span><a href="http://primal.zibraproject.org" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://primal.zibraproject.or</span></a><span>g/, described here </span><a href="https://www.nature.com/articles/nprot.2017.066" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.nature.com/articles/nprot.2017.066</span></a><span style = ":;">.</span></div><div class = "text-block"><span>Primer sequences are here: </span><a href="https://docs.google.com/spreadsheets/d/1M5I_C56ZC8_2Ycgm9EFieVlVNqxsP7dXAnGoBZy3nDo/edit?usp=sharing" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://docs.google.com/spreadsheets/d/1M5I_C56ZC8_2Ycgm9EFieVlVNqxsP7dXAnGoBZy3nDo/edit?usp=sharing</span></a></div><div class = "text-block"><span>The primer scheme .bed and .tsv files necessary for the ARTIC variant calling pipeline are at Zenodo: </span><a href="https://zenodo.org/record/3897530#.Xv5EFpMzadY" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://zenodo.org/record/3897530#.Xv5EFpMzadY</span></a></div><div class = "text-block">Version history: </div><div class = "text-block">V4: updated .bed and .tsv file link to point to Zenodo (and not google drive). </div><div class = "text-block">V1-V3: included primers sequences in the protocol, fixed step 17.12 from elute in "molecular grade water or Elution buffer" to elute in "10 mM Tris-HCl pH 8.0 with 50 mM NaCl", as suggested on the Oxford Nanopore protocol, changed images from ARTIC protocol image to our own. </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. It is good practice to carry a negative control (e.g. water) through the entire process from cDNA preparation to sequencing.
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 Snap cool in a prechilled metal rack or on ice
65 °C
A quick cooling step using a PCR cooling block or ice helps to inhibit secondary structure formation and can decrease variation in overall coverage.
?. [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 in a preheated PCR machine:Hold at
42 °C
70 °C
5 °C
?. [Primer pool preparation]
If required, resuspend lyophilised primers at a concentration of 100µM each
Primers for this protocol were designed using Primal Scheme and generate overlapping 1200bp amplicons. Primer names and dilutions are listed here: https://docs.google.com/spreadsheets/d/1M5I_C56ZC8_2Ycgm9EFieVlVNqxsP7dXAnGoBZy3nDo/edit?usp=sharing.We have tested multiplexing 1500 nt and 2000 nt amplicons as well, all work. These are included in the link. Here we will discuss just the protocol for 1200 nt amplicons as they worked best in our hands. You can order these as an oligo pool from IDT: https://sg.idtdna.com/site/order/poolentry/
?. [Primer pool preparation]
Primers used to generate 1200 bp amplicons are here:   ABCDEF1Primer
  NameSequencePoolLengthTmStart2SARSCoV_1200_1_LEFTACCAACCAACTTTCGATCTCTTGT12460.69303SARSCoV_1200_1_RIGHTGGTTGCATTCATTTGGTGACGC12261.4912054SARSCoV_1200_3_LEFTGGCTTGAAGAGAAGTTTAAGGAAGGT12661.1921535SARSCoV_1200_3_RIGHTGATTGTCCTCACTGCCGTCTTG12261.532576SARSCoV_1200_5_LEFTACCTACTAAAAAGGCTGGTGGC12260.5541677SARSCoV_1200_5_RIGHTAGCATCTTGTAGAGCAGGTGGA12261.1653598SARSCoV_1200_7_LEFTACCTGGTGTATACGTTGTCTTTGG12460.862839SARSCoV_1200_7_RIGHTGCTGAAATCGGGGCCATTTGTA12261.53740110SARSCoV_1200_9_LEFTAGAAGTTACTGGCGATAGTTGTAATAACT12960.59825311SARSCoV_1200_9_RIGHTTGCTGATATGTCCAAAGCACCA12260.29940012SARSCoV_1200_11_LEFTAGACACCTAAGTATAAGTTTGTTCGCA12760.741034313SARSCoV_1200_11_RIGHTGCCCACATGGAAATGGCTTGAT12261.81146914SARSCoV_1200_13_LEFTACCTCTTACAACAGCAGCCAAAC12361.551245015SARSCoV_1200_13_RIGHTCGTCCTTTTCTTGGAAGCGACA12261.381362116SARSCoV_1200_15_LEFTTTTTAAGGAATTACTTGTGTATGCTGCT12860.061454017SARSCoV_1200_15_RIGHTACACACAACAGCATCGTCAGAG12261.121573518SARSCoV_1200_17_LEFTTCAAGCTTTTTGCAGCAGAAACG12361.281662419SARSCoV_1200_17_RIGHTCCAAGCAGGGTTACGTGTAAGG12261.191775420SARSCoV_1200_19_LEFTGGCACATGGCTTTGAGTTGACA12261.911859621SARSCoV_1200_19_RIGHTCCTGTTGTCCATCAAAGTGTCCC12361.621967822SARSCoV_1200_21_LEFTTCTGTAGTTTCTAAGGTTGTCAAAGTGA12860.582055323SARSCoV_1200_21_RIGHTGCAGGGGGTAATTGAGTTCTGG12260.952164224SARSCoV_1200_23_LEFTACTTTAGAGTCCAACCAACAGAATCT12660.182251125SARSCoV_1200_23_RIGHTTGACTAGCTACACTACGTGCCC12261.522363126SARSCoV_1200_25_LEFTTGCTGCTACTAAAATGTCAGAGTGT12560.512463327SARSCoV_1200_25_RIGHTCATTTCCAGCAAAGCCAAAGCC12261.452579028SARSCoV_1200_27_LEFTTGGATCACCGGTGGAATTGCTA12261.752674429SARSCoV_1200_27_RIGHTTGTTCGTTTAGGCGTGACAAGT12260.742789430SARSCoV_1200_29_LEFTTGAGGGAGCCTTGAATACACCA12261.12867731SARSCoV_1200_29_RIGHTTAGGCAGCTCTCCCTAGCATTG12261.6129790  ABCDEF1Primer
  NameSequencePoolLengthTmStart2SARSCoV_1200_2_LEFTCCATAATCAAGACTATTCAACCAAGGGT22861.2711003SARSCoV_1200_2_RIGHTACAGGTGACAATTTGTCCACCG22261.3322664SARSCoV_1200_4_LEFTGGAATTTGGTGCCACTTCTGCT22261.6631445SARSCoV_1200_4_RIGHTCCTGACCCGGGTAAGTGGTTAT22261.4942626SARSCoV_1200_6_LEFTACTTCTATTAAATGGGCAGATAACAACTG22960.1852577SARSCoV_1200_6_RIGHTGATTATCCATTCCCTGCGCGTC22261.7563808SARSCoV_1200_8_LEFTCAATCATGCAATTGTTTTTCAGCTATTTTG23060.3972989SARSCoV_1200_8_RIGHTTGACTTTTTGCTACCTGCGCAT22261.39838510SARSCoV_1200_10_LEFTTTTACCAGGAGTTTTCTGTGGTGT22460.32930311SARSCoV_1200_10_RIGHTTGGGCCTCATAGCACATTGGTA22261.51045112SARSCoV_1200_12_LEFTATGGTGCTAGGAGAGTGTGGAC22261.481137213SARSCoV_1200_12_RIGHTGGATTTCCCACAATGCTGATGC22260.481256014SARSCoV_1200_14_LEFTACAGGCACTAGTACTGATGTCGT22361.121350915SARSCoV_1200_14_RIGHTGTGCAGCTACTGAAAAGCACGT22261.941464116SARSCoV_1200_16_LEFTACAACACAGACTTTATGAGTGTCTCT22660.181560817SARSCoV_1200_16_RIGHTCTCTGTCAGACAGCACTTCACG22261.171672018SARSCoV_1200_18_LEFTGCACATAAAGACAAATCAGCTCAATGC22762.031762219SARSCoV_1200_18_RIGHTTGTCTGAAGCAGTGGAAAAGCA22260.681870620SARSCoV_1200_20_LEFTACAATTTGATACTTATAACCTCTGGAACAC23060.151957421SARSCoV_1200_20_RIGHTGATTAGGCATAGCAACACCCGG22261.392069822SARSCoV_1200_22_LEFTGTGATGTTCTTGTTAACAACTAAACGAACA23061.442153223SARSCoV_1200_22_RIGHTAACAGATGCAAATCTGGTGGCG22262.032261224SARSCoV_1200_24_LEFTGCTGAACATGTCAACAACTCATATGA22660.132351825SARSCoV_1200_24_RIGHTATGAGGTGCTGACTGAGGGAAG22261.742473626SARSCoV_1200_26_LEFTGCCTTGAAGCCCCTTTTCTCTA22260.292569027SARSCoV_1200_26_RIGHTAATGACCACATGGAACGCGTAC22261.52685728SARSCoV_1200_28_LEFTTTTGTGCTTTTTAGCCTTTCTGCT22460.142778429SARSCoV_1200_28_RIGHTGTTTGGCCTTGTTGTTGTTGGC22261.8229007
ABCDEF1Primer
  NameSequencePoolLengthTmStart2SARSCoV_1200_1_LEFTACCAACCAACTTTCGATCTCTTGT12460.69303SARSCoV_1200_1_RIGHTGGTTGCATTCATTTGGTGACGC12261.4912054SARSCoV_1200_3_LEFTGGCTTGAAGAGAAGTTTAAGGAAGGT12661.1921535SARSCoV_1200_3_RIGHTGATTGTCCTCACTGCCGTCTTG12261.532576SARSCoV_1200_5_LEFTACCTACTAAAAAGGCTGGTGGC12260.5541677SARSCoV_1200_5_RIGHTAGCATCTTGTAGAGCAGGTGGA12261.1653598SARSCoV_1200_7_LEFTACCTGGTGTATACGTTGTCTTTGG12460.862839SARSCoV_1200_7_RIGHTGCTGAAATCGGGGCCATTTGTA12261.53740110SARSCoV_1200_9_LEFTAGAAGTTACTGGCGATAGTTGTAATAACT12960.59825311SARSCoV_1200_9_RIGHTTGCTGATATGTCCAAAGCACCA12260.29940012SARSCoV_1200_11_LEFTAGACACCTAAGTATAAGTTTGTTCGCA12760.741034313SARSCoV_1200_11_RIGHTGCCCACATGGAAATGGCTTGAT12261.81146914SARSCoV_1200_13_LEFTACCTCTTACAACAGCAGCCAAAC12361.551245015SARSCoV_1200_13_RIGHTCGTCCTTTTCTTGGAAGCGACA12261.381362116SARSCoV_1200_15_LEFTTTTTAAGGAATTACTTGTGTATGCTGCT12860.061454017SARSCoV_1200_15_RIGHTACACACAACAGCATCGTCAGAG12261.121573518SARSCoV_1200_17_LEFTTCAAGCTTTTTGCAGCAGAAACG12361.281662419SARSCoV_1200_17_RIGHTCCAAGCAGGGTTACGTGTAAGG12261.191775420SARSCoV_1200_19_LEFTGGCACATGGCTTTGAGTTGACA12261.911859621SARSCoV_1200_19_RIGHTCCTGTTGTCCATCAAAGTGTCCC12361.621967822SARSCoV_1200_21_LEFTTCTGTAGTTTCTAAGGTTGTCAAAGTGA12860.582055323SARSCoV_1200_21_RIGHTGCAGGGGGTAATTGAGTTCTGG12260.952164224SARSCoV_1200_23_LEFTACTTTAGAGTCCAACCAACAGAATCT12660.182251125SARSCoV_1200_23_RIGHTTGACTAGCTACACTACGTGCCC12261.522363126SARSCoV_1200_25_LEFTTGCTGCTACTAAAATGTCAGAGTGT12560.512463327SARSCoV_1200_25_RIGHTCATTTCCAGCAAAGCCAAAGCC12261.452579028SARSCoV_1200_27_LEFTTGGATCACCGGTGGAATTGCTA12261.752674429SARSCoV_1200_27_RIGHTTGTTCGTTTAGGCGTGACAAGT12260.742789430SARSCoV_1200_29_LEFTTGAGGGAGCCTTGAATACACCA12261.12867731SARSCoV_1200_29_RIGHTTAGGCAGCTCTCCCTAGCATTG12261.6129790
ABCDEF1Primer
  NameSequencePoolLengthTmStart2SARSCoV_1200_2_LEFTCCATAATCAAGACTATTCAACCAAGGGT22861.2711003SARSCoV_1200_2_RIGHTACAGGTGACAATTTGTCCACCG22261.3322664SARSCoV_1200_4_LEFTGGAATTTGGTGCCACTTCTGCT22261.6631445SARSCoV_1200_4_RIGHTCCTGACCCGGGTAAGTGGTTAT22261.4942626SARSCoV_1200_6_LEFTACTTCTATTAAATGGGCAGATAACAACTG22960.1852577SARSCoV_1200_6_RIGHTGATTATCCATTCCCTGCGCGTC22261.7563808SARSCoV_1200_8_LEFTCAATCATGCAATTGTTTTTCAGCTATTTTG23060.3972989SARSCoV_1200_8_RIGHTTGACTTTTTGCTACCTGCGCAT22261.39838510SARSCoV_1200_10_LEFTTTTACCAGGAGTTTTCTGTGGTGT22460.32930311SARSCoV_1200_10_RIGHTTGGGCCTCATAGCACATTGGTA22261.51045112SARSCoV_1200_12_LEFTATGGTGCTAGGAGAGTGTGGAC22261.481137213SARSCoV_1200_12_RIGHTGGATTTCCCACAATGCTGATGC22260.481256014SARSCoV_1200_14_LEFTACAGGCACTAGTACTGATGTCGT22361.121350915SARSCoV_1200_14_RIGHTGTGCAGCTACTGAAAAGCACGT22261.941464116SARSCoV_1200_16_LEFTACAACACAGACTTTATGAGTGTCTCT22660.181560817SARSCoV_1200_16_RIGHTCTCTGTCAGACAGCACTTCACG22261.171672018SARSCoV_1200_18_LEFTGCACATAAAGACAAATCAGCTCAATGC22762.031762219SARSCoV_1200_18_RIGHTTGTCTGAAGCAGTGGAAAAGCA22260.681870620SARSCoV_1200_20_LEFTACAATTTGATACTTATAACCTCTGGAACAC23060.151957421SARSCoV_1200_20_RIGHTGATTAGGCATAGCAACACCCGG22261.392069822SARSCoV_1200_22_LEFTGTGATGTTCTTGTTAACAACTAAACGAACA23061.442153223SARSCoV_1200_22_RIGHTAACAGATGCAAATCTGGTGGCG22262.032261224SARSCoV_1200_24_LEFTGCTGAACATGTCAACAACTCATATGA22660.132351825SARSCoV_1200_24_RIGHTATGAGGTGCTGACTGAGGGAAG22261.742473626SARSCoV_1200_26_LEFTGCCTTGAAGCCCCTTTTCTCTA22260.292569027SARSCoV_1200_26_RIGHTAATGACCACATGGAACGCGTAC22261.52685728SARSCoV_1200_28_LEFTTTTGTGCTTTTTAGCCTTTCTGCT22460.142778429SARSCoV_1200_28_RIGHTGTTTGGCCTTGTTGTTGTTGGC22261.8229007
?. [Primer pool preparation]
If you have ordered each primer independently and need to generate primer pool stocks: add   of each primer from Pool 1 to a  Eppendorf labeled “Pool 1 (100µM)” and each primer from Pool 2 to a  Eppendorf labelled  “Pool 2 (100µM)”. 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 (1200 nt amplicons),  pool 1 has 30 primers and pool 2 has 28 primers, so the requirement is 1.13µL for primer pool 1 and 1.05µL for primer pool 2 (10uM) per 25µL reaction. However, as these values are relatively close, we round up and down to 1.1ul for both pools, so the pools can be made in a similar fashion. 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            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
1.1 µl
1.1 µl
15.9 µl
15.9 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 and Extension             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. We typically use 30 cycles.
Final concentrations of PCR products can range from ~20- 150ng/ul.
?. [Pooling and PCR quantification]
Label a  Eppendorf tube for each sample and combine the two pools the PCR reaction as follows:Component                                        VolumePool 1 PCR reaction                       Pool 2 PCR reaction                       Total
1.5 mL
25 µl
25 µl
50 µl
At this stage, care should be taken with amplified PCR products. Only open tubes in a designated post-PCR workspace with equipment that is separate from areas where primers and mastermixes are handled. After combining the two pools of amplified DNA, the PCR products can be used for Oxford Nanopore Sequencing, using the RAPID barcode kit RBK004, as described in this protocol (below, Steps 15 onward). Alternatively, these amplicons can be used for Oxford Nanopore Sequencing, following Josh Quick's ligation based protocol (CoV-2019 sequencing protocol v2, dx.doi.org/10.17504/protocols.io.bdp7i5rn, at step 15)  using the SQK-LSK109 kit.Alternatively, these amplicons can also be used for Illumina sequencing, such as found here: x.doi.org/10.17504/protocols.io.betejeje We have found that performing an Ampure XP bead clean up at this stage does not improve performance. Therefore, it is not necessary to clean up the PCR reaction at this step.
?. [Pooling and PCR quantification]
Quantify DNA using a Qubit or other method. Quantification using Nanodrop is not recommended.
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?. [Normalisation]
Label a   PCR tube for each sample.
0.2 mL
?. [Normalisation]
Adjust the amount of DNA in the tube to be  total per sample in  molecular grade water. For example if your PCR reaction is at 100ng/ul, add 1ul of the PCR reaction to 6.5ul of molecular grade water. Input to the Rapid Barcoding kit will vary depending on the amplicon length but we have determined 50-200 ng works for efficient barcoding of this amplicon length.  Use 7.5ul of the negative control, even if there is no detectable DNA in the PCR reaction.
100 ng
7.5 µl
?. [Rapid barocoding using the SQK RBK004]
Mulitple samples can be run on the same flow cell by barcoding. Up to 12 samples at a time can be run. Amplicons from each sample will be individually barcoded in the following steps. These follow the RBK004 protocol from Oxford Nanopore. It is highly recommended to use their protocol for the following steps.  Tip: aliquot the Rapid barcodes into a PCR strip to enable multichannelling.
?. [Rapid barocoding using the SQK RBK004]
Add  of each diluted PCR reaction from step 15 to the labeled PCR tube.  Set up the following reaction for each sample:Component                                                                                                            VolumeDNA amplicons from step 15  (100ng total)                                                         Fragmentation Mix RB01-12 (one for each sample, included in kit)                 Total
7.5 µl
7.5 µl
2.5 µl
10 µl
?. [Rapid barocoding using the SQK RBK004]
Mix gently by flicking the tube, and spin down.
?. [Rapid barocoding using the SQK RBK004]
Incubate the reaction in a PCR machine:     for          for              for
30 °C
80 °C
4 °C
?. [Rapid barocoding using the SQK RBK004]
Pool all barcoded samples, noting the total volume.
?. [Rapid barocoding using the SQK RBK004]
Ampure XP Bead Cleanup. Use a 1:1 ratio of sample to beads.
?. [Rapid barocoding using the SQK RBK004]
Add of RAP (from the RBK004 kit)  to cleaned, barcoded DNA from step 17 . Mix gently by flicking the tube, and spin down.
1 µl
10 µl
?. [Rapid barocoding using the SQK RBK004]
Incubate the reaction for  at room temperature.
?. [Rapid barocoding using the SQK RBK004]
The prepared library is used for loading into the MinION flow cell according to Oxford Nanopore Rapid Barcoding (RBK004) protocol. Please refer to the Oxford Nanopore Rapid Barcoding RBK004 protocol at this stage. Store the library on ice until ready to load.
?. [MinION sequencing]
Start the sequencing run using MinKNOW.
?. [MinION sequencing]
Depending on the variation in coverage of each amplicon, generally, you will need approx 10,000 to 20,000 reads or 10-20Mb per sample to confidently assemble and call variants. This can typically be achieved on a minION flow cell in under two hours when runnning 12 samples. Shorter, if running fewer samples.
?. [MinION sequencing]
The primer scheme .bed and .tsv files necessary for the ARTIC variant calling pipeline are at Zenodo: https://zenodo.org/record/3897530#.Xv5EFpMzadY