Apr 29, 2020
Version 1
SARS-CoV-2 Sequencing on Illumina MiSeq Using ARTIC Protocol: Part 1 - Tiling PCR V.1
- Joel Sevinsky1,
- Arian Nassiri2,
- Erin Young3,
- Heather Blankenship4,
- Kevin Libuit2,
- Kelly Oakeson3,
- Lauren Turner2,
- StaPH-B Consortium5
- 1Theiagen Genomics;
- 2Virginia Division of Consolidated Laboratory Services;
- 3Utah Public Health Laboratory;
- 4Michigan Department of Health and Human Services;
- 5State Public Health Bioinformaticians
- Coronavirus Method Development Community
- StaPH-B
External link: https://doi.org/10.1093/ve/veac104
Protocol Citation: Joel Sevinsky, Arian Nassiri, Erin Young, Heather Blankenship, Kevin Libuit, Kelly Oakeson, Lauren Turner, StaPH-B Consortium 2020. SARS-CoV-2 Sequencing on Illumina MiSeq Using ARTIC Protocol: Part 1 - Tiling PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bfefjjbn
Manuscript citation:
Halfmann PJ, Minor NR, III LAH, Maddox R, Moreno GK, Braun KM, Baker DA, Riemersa KK, Prasad A, Alman KJ, Lambert MC, Florek K, Bateman A, Westergaard R, Safdar N, Andes DR, Kawaoka Y, Fida M, Yao JD, Friedrich TC, O’Connor DH (2022) Evolution of a globally unique SARS-CoV-2 Spike E484T monoclonal antibody escape mutation in a persistently infected, immunocompromised individual. Virus Evolution 9(2). doi: 10.1093/ve/veac104
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol. Comments and feedback appreciated.
Created: April 21, 2020
Last Modified: April 29, 2020
Protocol Integer ID: 36007
Abstract
This protocol is an adaption of several circulating protocols on SARS-CoV-2 sequencing using the ARTIC protocol. Its purpose is to simplify things for the average state public health laboratory, using equipment and expertise they currently posess, most likely from their funded PulseNet activities.
This protocol is derived from other works, including:
pcr-tiling-ncov-PTC_9096_v109_revE_06Feb2020-minion.pdf Materials
STEP MATERIALS
DNA LoBind 1.5mL microcentrifuge tubesFisher ScientificCatalog #13-698-791
AMPure XP Beckman CoulterCatalog #A63881
Random primer mixNew England BiolabsCatalog #S1330S
Deoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
SuperScript™ IV Reverse TranscriptaseThermo FisherCatalog #18090010
DNA LoBind 1.5mL microcentrifuge tubesFisher ScientificCatalog #13-698-791
96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Q5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
RT-PCR Grade WaterThermo FisherCatalog #AM9935
Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Protocol materials
AMPure XP Beckman CoulterCatalog #A63881
Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Random primer mixNew England BiolabsCatalog #S1330S
Deoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
DNA LoBind 1.5mL microcentrifuge tubesFisher ScientificCatalog #13-698-791
Q5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
DNA LoBind 1.5mL microcentrifuge tubesFisher ScientificCatalog #13-698-791
SuperScript™ IV Reverse TranscriptaseThermo FisherCatalog #18090010
96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
RT-PCR Grade WaterThermo FisherCatalog #AM9935
Random primer mixNew England BiolabsCatalog #S1330S
Deoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
SuperScript™ IV Reverse TranscriptaseThermo FisherCatalog #18090010
DNA LoBind 1.5mL microcentrifuge tubesFisher ScientificCatalog #13-698-791
96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504
Q5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
RT-PCR Grade WaterThermo FisherCatalog #AM9935
DNA LoBind 1.5mL microcentrifuge tubesFisher ScientificCatalog #13-698-791
AMPure XP Beckman CoulterCatalog #A63881
Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
ARTIC Protocol - Prepping Nucleic Acid
ARTIC Protocol - Prepping Nucleic Acid
Getting RNA ready for cDNA Creation
In this section we cover the process of preparing your nucleic acid/RNA extractions, from your qPCR diagnostic test, to be used in viral sequencing. You will need to have Ct values for each of your specimens because this will determine your dilution factor prior to starting your cDNA preparation. Dilute your sample based on the chart below:
| qPCR Ct** | Dilution Factor | |
| 18-35 | none | |
| 15-18 | 1:10 | |
| 12-15 | 1:100 |
Dilution factor guide
You can use the attached worksheet to help with sample organization:Initial Sample Dilution Sheet.pdf 
Initial Sample Dilution Sheet.xlsx
Initial Sample Dilution Sheet.pdf Initial Sample Dilution Sheet.xlsx Note
**NOTE: If you do not have Ct values from your diagnostic test, you can use the RNA extractions without dilution. An alternative approach that has been successful that helps with througput is to use every samples undiluted, and only if it fails to sequence correctly go back and dilute. Some labs have been reporting that greater than 90% of their specimens will sequence fine undiluted.
ARTIC Protocol - cDNA Preparation - Reverse Transcription
ARTIC Protocol - cDNA Preparation - Reverse Transcription
cDNA/Reverse Transcription Section Date/Initials:_________________
In this section we cover the process of taking your nucleic acid extraction from your qPCR diagnostic test and use it as starting material for the sequencing.
[ ] In a PCR hood, mix the following reagents and add to a 0.2 mL PCR tube on a cold block plus 11 µL of RNA sample:
| Reagent | Volume (uL) | MM for N+2 samples | |
| 60 uM random hexamers and anchored polyT(23) | 1.0 | ||
| 10mM dNTPs | 1.0 | ||
| Total | 2.0 |
Master mix calculations
Each reaction should have 13 µL when mixed. If using master mix, it is recommended to add the 2 µL of the master mix to the PCR tube first, then add the 11 µL of RNA to help prevent contamination.
Random primer mixNew England BiolabsCatalog #S1330S
Lot# _______________ Exp. Date _______________
Deoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
Lot# _______________ Exp. Date _______________
[ ] Mix gently, spin down, and return toOn ice .
[ ] Preheat Thermocycler to65 °C , with heated lid at 105 °C
[ ] Incubate the reaction at 65 °C for 00:05:00 , followed by an immediate snap-cool on On ice for at least00:01:00 .
[ ] In a clean 1.5 mL LoBind tube (96 well plates can also be used) on On ice , mix together the following reagents:
| Reagent | Volume (uL) | MM for N+2 samples | |
| SuperScript IV RT 5X Buffer** | 4.0 | ||
| 100mM DTT** | 1.0 | ||
| RNaseOUT RNase inhibitor | 1.0 | ||
| Superscript IV Reverse Transcriptase** | 1.0 | ||
| Total | 7.0 |
Master mix for RT reaction.
Note
**Note: All these reagents are part of the SuperScript IV Reverse Transcriptase kit.
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Lot# _______________ Exp. Date _______________
SuperScript™ IV Reverse TranscriptaseThermo FisherCatalog #18090010
Lot# _______________ Exp. Date _______________
DNA LoBind 1.5mL microcentrifuge tubesFisher ScientificCatalog #13-698-791
96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504
[ ] After the RNA sample has cooled for at least 00:01:00 , longer if needed to make master mix, add 7 µL of the above master mix to the sample.
[ ] Mix gently by flicking, and spin down. Return tube to On ice .
[ ] Preheat thermocycler to 42 °C , with heated lid at 105 °C
[ ] Incubate sample using the following COVID WGS Reverse Transcription program:
| Step | Temp | Time | Cycle | |
| Reverse Transcription | 42 C | 50:00 | 1 | |
| RT Inactivation | 70 C | 10:00 | 1 | |
| Cool | 4 C | Hold | Hold |
SARS-CoV-2 Reverse Transcription Program
ARTIC Protocol - Tiled PCR Section
ARTIC Protocol - Tiled PCR Section
Tiled PCR Section Date/Initials:_________________
This section outlines the process for the tiled PCR approach from the ARTIC protocol. A seperate document will be provided outlining how to order primers and make the two different primer pools needed for this section. Hopefully most first time labs will receive aliquots of both Pool A and Pool B from labs that have successfully completed this protocol before to help with any potential troubleshooting.
[ ] Set up two individual reactions using primer pool A and primer pool B in 0.2 mL PCR tubes according to the following table:
| Reagent | Pool A (uL) | MM for N+2 samples | Pool B (uL) | MM for N+2 samples | |
| Q5 Hot Start HiFi 2x MM | 12.5 | 12.5 | |||
| Primer pool at 10uM (A or B)** | 3.7 | 3.7 | |||
| Nuclease-free water | 6.3 | 6.3 | |||
| Total | 22.5 | 22.5 |
Master Mix for Tiled PCR
Note
**See protocol on primer design for SARS-CoV-2. If this is your first attempt, it would be best to receive aliquots of the primers from a lab that has successfully sequenced SARS-CoV-2 first.
Q5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
Lot# _______________ Exp. Date _______________
RT-PCR Grade WaterThermo FisherCatalog #AM9935
Lot# _______________ Exp. Date _______________
Note
Any PCR grade water will do in this step. Not necessary to use the reagent listed.
[ ] Add 2.5 µL sample cDNA to each pool.
[ ] Mix gently and spin down prior to loading on the thermocycler.
[ ] Run the following thermocycler program:
| Step | Temp | Time | Cycles | |
| Initial Denaturation | 98°C | 0:30 | 1 | |
| Denaturation | 98°C | 0:15 | 25 or 35** | |
| Anneal and Extension | 65°C | 5:00 | 25 or 35** | |
| Cool | 4°C | Hold | Hold |
SARS-CoV-2 Tiled PCR - see note below.
Note
**Note: If starting RNA samples had a qPCR Ct value in the range of 12-21, use 25 cycles.
If starting RNA samples had a qPCR Ct values in the range of 21-35, use 35 cycles. An alternative approach that has been successful that helps with througput is to run every specimen undiluted and for 35 cycles, and only if it fails to sequence correctly go back and dilute and/or run for fewer cycles. Some labs have been reporting that greater than 90% of their specimens will sequence fine undiluted for 35 cycles.
ARTIC Protocol - Clean-Up and Size Selection
ARTIC Protocol - Clean-Up and Size Selection
Section for Clean-Up and Size Selection Date/Initials:_________________
This process is similar to the bead clean-ups performed for the PulseNet WGS protocol. The same beads and magnets may be used, although it is recommended to have seperate beads to help prevent contamination.
[ ] Combine the 25 µL reaction from Pool A and the25 µL reaction from Pool B into a new 1.5 mL LoBind tube. One tube per sample.
DNA LoBind 1.5mL microcentrifuge tubesFisher ScientificCatalog #13-698-791
[ ] Re-suspend AMPure XP beads by vortexing.
AMPure XP Beckman CoulterCatalog #A63881
Lot# _______________ Exp. Date _______________
Note
The AMPure XP is available in 5 mL , 60 mL , and 450 mL sizes. Please choose the appropriate size for your throughput.
[ ] Add 50 µL of re-suspended AMPure XP beads to the reaction and mix.
[ ] Incubate on a rotator mixer for 00:10:00 at Room temperature .
[ ] Prepare 500 µL 80 % volume ethanol using the following calculation:
Sample# + 1: ________________
0.5ml x Sample# = ________ mL total volume
mL total volume x 0.8 = ________ mL EtOH
Total volume _______mL - _______mL EtOH = ________mL H2O
[ ] Spin down sample and pellet the beads on a magnet** for approximately 00:05:00 . Keep tubes on the magnet** and pipette off supernatant.
Note
**Use the magnetic stands from your PulseNet protocols.
[ ] While on the magnet, wash beads with 200 µL freshly prepared 80 % volume EtOH without disturbing the pellet. Rotate the tube to allow the bead pellet to migrate towards the opposite side of the tube. Remove EtOH
[ ] Repeat previous step.
[ ] Spin down and place the tubes back on the magnet. Pipette off any residual ethanol and allow to dry for approximately 00:00:30 . Take care to not over dry the pellet.
[ ] Remove tubes from the magnet and re-suspend pellet in 30 µL of nuclease-free water**.
Note
**Use whatever nuclease free water was used in previous steps.
[ ] Incubate atRoom temperature for approximately 00:02:00 .
[ ] Pellet the beads on a magnet until eluate is clear and colorless
[ ] Remove ~30 µL of eluate and place in a clean 1.5 mL LoBind tube.
[ ] Quantify eluted sample on Qubit fluorometer or similar instrument and store completed PCR amplified cDNA prep at-20 °C .
Equipment
Qubit Fluorometer
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
LINK
Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Note
Earlier versions of the Qubit, or any other method for accurate DNA quantitation can be used. Most labs should have this equipment available in their NGS sections.
[ ] PCR amplified cDNA is now ready for Illumina library preparation. Please proceed to Part 2 for DNA Flex Library Preparation.
Note
NOTE: For those of you familiar with the PulseNet protocols for WGS of bacterial pathogens, you are at the equivalent stage where you have extracted you DNA from your colony and are ready to begin library preparation, usually with DNA Flex.