TITLE: SARS-CoV2 genome sequencing protocol (1200bp amplicon "midnight" primer set, using Nanopore Rapid kit)
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.   </div><br/><div class = "text-block"><span>UPDATE: The Rapid barcoding protocol is now (March 2021) available on the Oxford Nanopore website for 1-96 samples "PCR tiling of SARS-CoV-2 virus with rapid barcoding (SQK-RBK110.96)" </span><a href="https://community.nanoporetech.com/protocols/pcr-tiling-of-sars-cov-2-virus-with-rapid-barcoding-sqk-rbk110/v/PCTR_9125_v110_revA_24Mar2021" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://community.nanoporetech.com/protocols/pcr-tiling-of-sars-cov-2-virus-with-rapid-barcoding-sqk-rbk110/v/PCTR_9125_v110_revA_24Mar2021</span></a></div><br/><div class = "text-block">This is a modification of the ARTIC amplicon V3 sequencing protocol for MinION for nCoV-2019 developed by Josh Quick. The ARTIC method uses 400 base pair amplicons and the Oxford Nanopore Ligation barcoding kit (LSK-109). </div><br/><div class = "text-block">The protocol described here, dubbed the Midnight protocol, makes two important changes to the ARTIC V3 protocol: </div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"> We have increased the size of the amplicon size to 1200bp. Using just 29 amplicons, the midnight primers anneal to only 4.5% of the viral genome compared to 17% or more of the genome, lowering the chance that viral mutations will cause dropouts due to primer mismatches. </li><li style = "counter-reset:ol0;">We use the RAPID barcode kit RBK004  (or, available as of March 2021 for 1-96 samples with the RBK110-96), which requires less time and fewer reagents than the ligation based LSK-109 protocol.</li></ol></div><br/><div class = "text-block"><span>The amplicons produced in this protocol can also be used for Illumina sequencing (i.e.  using a transposon based shearing like Nextera or the Lotus kit  </span><a href="https://sg.idtdna.com/pages/landing/coronavirus-research-reagents/ngs-assays#resources" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://sg.idtdna.com/pages/landing/coronavirus-research-reagents/ngs-assays#resources</span></a><span style = ":;">)</span></div><br/><div class = "text-block"><span>The original publication that describes this protocol is here along with important considerations is here:    </span><a href="https://academic.oup.com/biomethods/article/5/1/bpaa014/5873518?login=true" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://academic.oup.com/biomethods/article/5/1/bpaa014/5873518?login=true</span></a></div><br/><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><br/><div class = "text-block"><span>Primer sequences are here (and listed directly in the protocol)   </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><br/><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><br/><br/><div class = "text-block">Version history: </div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">V6: updated to indicate primers can be purchased from IDT and removed link to opools. </li><li style = "counter-reset:ol0;list-style-type:disc;"><span>V5: Changed Reverse transcription part of the protocol to use the LunaScript™ RT SuperMix Kit in place of SSIV as it is easier, performs similarly, and is less expensive. The PCR step was changed to use the Q5</span><span style = "vertical-align:super;">®</span><span>Hot Start High-Fidelity 2X Master Mix in place of the Q5 reagents individually, as this cuts down pipetting steps, performs similarly, and is similar in cost.</span></li><li style = "counter-reset:ol0;list-style-type:disc;">V4: updated .bed and .tsv file link to point to Zenodo (and not google drive). </li><li style = "counter-reset:ol0;list-style-type:disc;">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. </li></ul></div></div>

[STEPS]
?. [cDNA preparation]
UPDATE March 2021: This protocol is now listed on the Oxford Nanopore Protocols website for 1-24, 1-48 or 1-96 samples using the RBK110.96 kit as the Midnight protocol PCR tiling of SARS-CoV-2 virus with rapid barcoding (SQK-RBK110.96)" Mix the following components in an 0.2mL 8-strip tube on ice; Component                                        VolumeLunaScript®RT SuperMix Kit (5x)           Template RNA                                                                                        Total
2 µl
8 µl
10 µl
Viral RNA input from a clinical sample should be between Ct 18-35. If Ct is between 12-15, then you can 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.  You will only carry 5 µl of this reaction into the next PCR step.
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 in a PCR machine:Hold at
25 °C
55 °C
95 °C
4 °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. Primers are available as two pools from IDT, SARS-Cov2-Midnight-1200, 500rxn, 10007184  (or can be purchase individually and pooled manually).
?. [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 and volume per sample            Pool 1                          Pool 2Q5® Hot Start HF 2x Master Mix             Primer Pool 1 (10µM)                                Primer Pool 2 (10µM)                                Nuclease-free water                                                           Total
12.5 µl
12.5 µl
1.1 µl
0 µl
0 µl
1.1 µl
8.9 µl
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 labelled PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet. The PCR can also be done in half reaction size for cost savings.
?. [Multiplex PCR]
In the extraction and sample addition cabinet: add  each cDNA sample to a tube containing   Pool 1. Mix well by pipetting.Add  each cDNA sample to a tube containing   Pool 2. Mix well by pipetting.
2.5 µl
22.5 µl
2.5 µl
22.5 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
We generally use 32 cycles, however the original ARTIC protocol states "Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35".  We have found no improvements to the protocol by changing the annealing temperature. We found it is not advisable to reduce the extension time below 5 minutes.
Final concentrations of PCR products typically range from ~5 - 150ng/ul as measured by Qubit.
?. [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
10 µl
10 µl
20 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: https://www.protocols.io/view/sars-cov-2-genome-sequencing-using-long-pooled-amp-befyjbpw  or https://sg.idtdna.com/pages/landing/coronavirus-research-reagents/ngs-assays#resourcesWe have found that performing an Ampure XP bead clean up at this stage does not improve performance and omitting it can save costs. Therefore, we do not do a bead clean up step at this stage.
?. [Pooling and PCR quantification]
Quantify DNA using a Qubit or other method. Quantification using Nanodrop is not recommended. If you do not have a Qubit or want to save time/costs, you can omit this step.
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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. If there is under 100ng or you do not know the concentration, simply use all  of the pooled PCR reaction. Use the full of the negative control, even if there is no detectable DNA in the PCR reaction.
100 ng
7.5 µl
7.5 µl
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 using the RBK004 kit, up to 96  if you are using the RBK110.96 kit. Amplicons from each sample will be individually barcoded in the following steps. It is highly recommended to use the current Oxford Nanopore Protocol for the following steps.  Tip: aliquot the Rapid barcodes into a PCR strip to enable multi channelling. They are briefly described here.
?. [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 running 12 samples. Less time if running fewer samples. You can also use the Rampart pipeline for a real time assessment of the coverage, which enables you to turn off the sequencing when you have captured enough data. Details on that here:  https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html
?. [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