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# Goal/Experiment:
To identify and monitor SARS-CoV-2 variant evolution, using a surveillance sequencing protocol optimized for Oxford Nanopore PromethION.

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## A SARS-CoV-2 Surveillance Sequencing Protocol Optimized for Oxford Nanopore PromethION

**Authors**:
Jannatul Ferdous, Torri Weathers, Visva Bharati Barua, Erin Stiers, Adam France, Kevin C Lambirth, Cynthia Gibas, Jessica A Schlueter  
*UNC Charlotte*  

**Published**: October 15, 2021  
**DOI**: [dx.doi.org/10.17504/protocols.io.butbnwin](https://dx.doi.org/10.17504/protocols.io.butbnwin)  

---

### Introduction
To identify and monitor SARS-CoV-2 variant evolution, UNC Charlotte has introduced a surveillance sequencing program. This protocol, optimized for the Oxford Nanopore PromethION, prepares SARS-CoV-2 viral genome libraries for next-generation sequencing. The protocol is designed to work in a 96-well format, producing sufficient sequence data for genome assembly to meet GISAID and NCBI submission standards. It addresses isolate sequencing failures due to low viral titers and provides guidelines for cost-effective sequencing of high Cq value clinical samples.

---

### Safety and Preparations
- **Wear PPE** at all times.
- **Clean workbench** and pipettes with 70% ethanol before use.
- **Handle samples carefully** during SPRI clean-up, barcoding, and adapter ligation to avoid significant loss of beads.

---

### Reagents and Equipment
#### cDNA Synthesis
- **ABI HC cDNA kit** (Fisher cat #4368813)
- **10x RT Buffer** (ABI HC cDNA kit, Fisher cat #4368813)
- **10x RT Random primers** (ABI HC cDNA kit, Fisher cat #4368813)
- **25x dNTPs Mix**, 100 mM
- **Multiscribe RT**, 50 U/µL (ABI HC cDNA kit, Fisher cat #4368813)
- **MgCl2**, 50 mM

#### ARTIC PCR Amplification
- **Q5 Reaction Buffer** (New England Biolabs, cat #M0491L)
- **Q5 High Fidelity Polymerase** (New England Biolabs, cat #M0491L)
- **ARTIC V3 primer pool 1** (IDT DNA, diluted to 10 µM)
- **ARTIC V3 primer pool 2** (IDT DNA, diluted to 10 µM)
- **2.5 mM dNTP Mix**

#### SPRI Clean-up
- **AMPure XP beads** (Beckman Coulter cat #A63881)
- **80% Ethanol**, fresh
- **Omega EB** (Omega BioTek, PD089)

#### Qubit Quantification
- **Invitrogen Qubit 1x dsDNA HS Assay Kit** (Fisher cat #Q33231)

#### Library End-Repair
- **Ultra II End-Prep Reaction Buffer**
- **Ultra II End-Prep Reaction Enzyme**

#### Sample Barcoding
- **Native Barcoding Expansion 96** (Oxford Nanopore, EXP-NBD196)
- **Ultra II Ligation Master Mix** (New England Biolabs cat #E7546L)
- **Ultra II Ligation Enhancer** (New England Biolabs cat #E7546L)
- **Short Fragment Buffer** (Oxford Nanopore, EXP-SFB001)
- **Elution Buffer** (EB)

#### Adapter Ligation
- **Adapter Mix II** (Oxford Nanopore, EXP-MRT001 or part of EXP-NBD196)
- **Quick T4 DNA Ligase** (New England Biolabs cat #E6056L)
- **Short Fragment Buffer** (Oxford Nanopore, EXP-SFB001)
- **Elution Buffer** (EB; Oxford Nanopore EXP-AUX001)

---

### Protocol

#### cDNA Synthesis

1. **Reagents:**
   - 10x RT Buffer
   - 50mM MgCl2
   - 10x RT Random Primer
   - 25x dNTPs Mix
   - Multiscribe RT

2. **Prepare Master Mix 1 (MM1) in a 1.5 mL Lobind tube on ice:**
   - *For 96 samples*  
     Nuclease Free Water: 424 µL  
     10x Random Primer: 212 µL  
     25x dNTPs Mix: 84.8 µL  

3. **Array RNA into Plate and Mix:**
   - *For Regular Samples:*  
     Array 10 µL RNA into a 96-well plate on an ice block. Add 6.8 µL of MM1 to each well.

   - *For High Cq Samples:*  
     Array 20 µL RNA and add 13.6 µL MM1 to each well.

   - *For Reduced Cost Samples:*  
     Array 5 µL RNA and add 3.4 µL MM1 to each well.

4. **Incubate Reaction:**
   - Seal plate, mix by quick spin. Incubate at 65°C for 5 minutes.

5. **Prepare Master Mix 2 (MM2) in a 1.5 mL Lobind tube on ice:**
   - *For 96 samples*  
     10x RT Buffer: 212 µL  
     Multiscribe RT: 106 µL  
     MgCl2: 21.2 µL  

6. **Add MM2 to Each Well:**
   - *For Regular Samples:* Add 3.2 µL MM2 to the RNA plate.
   - *For High Cq Samples:* Add 6.4 µL MM2 per well.
   - *For Reduced Cost Samples:* Add 1.6 µL MM2 per well.

7. **Incubate Further:**
   - Seal and mix by quick spin.  
   25°C for 10 minutes  
   37°C for 2 minutes  
   85°C for 5 minutes  
   Hold at 4°C

8. **Store Plate:** 
   - For long term: -20°C  
   - Short term: 4°C

#### ARTIC PCR Amplification

9. **Reagents:**
   - 5x Q5 Reaction Buffer
   - Q5 High Fidelity Polymerase
   - V3 ARTIC primer pool 1 (diluted to 10 µM)
   - V3 ARTIC primer pool 2 (diluted to 10 µM)

10. **PCR Plate Setup:**
    - **ARTIC Pool 1:**  
      Master Mix:  
      Nuclease-free water: 927.5 µL  
      Reaction Buffer: 530 µL  
      V3 Pool 1: 424 µL  
      2.5mM dNTP mix: 212 µL  
      Q5 High fidelity polymerase: 26.5 µL  

    - **ARTIC Pool 2:**  
      Master Mix:  
      Nuclease-free water: 927.5 µL  
      Reaction Buffer: 530 µL  
      V3 Pool 2: 424 µL  
      2.5mM dNTP mix: 212 µL  
      Q5 High fidelity polymerase: 26.5 µL

11. **Distribute PCR Components for ARTIC Pools 1 & 2:**
    - Mix 20 µL MM1 into the ARTIC Pool 1 plate and add 5 µL of cDNA.
    - Mix 20 µL MM2 into the ARTIC Pool 2 plate and add 5 µL of cDNA.

12. **Set PCR Cycling Program:**
    - 1 Cycle: 98°C for 30 seconds  
    - 20 Cycles: 94°C for 16 seconds → 65°C for 5 minutes → 94°C for 16 seconds → 65°C for 5 minutes  
    - Hold at 4°C.

    **Store at**  
    - Long term: -20°C  
    - Short term: 4°C

#### SPRI Clean-Up

13. **Reagents:**
    - AMPure XP Beads
    - 80% Ethanol Fresh
    - Omega EB

14. **Prepare Fresh 80% Ethanol:**
    - Mix 40 mL 100% Ethanol with 10 mL H2O in a 50 mL tube.

15. **Clean-up Procedure:**
    - Spin PCR Product Plates.
    - Transfer and mix AMPure Beads.
    - Incubate 15 minutes.
    - Wash with 80% ethanol.
    - Elute DNA with 33 µL Omega EB.

#### Qubit Quantification

16. **Reagents:**
    - Qubit Quantification Kit (dsDNA HS Assay)
    - Standards (Included in Kit)

17. **Qubit reagent preparation:**
    - dsDNA HS Buffer: 24278 µL
    - Qubit Dye: 122 µL
    - Mix well, vortex and spin.

18. **Run Qubit Assay Using Standard Protocols:**

#### Library End-Repair

19. **Reagents:**
    - NEB Ultra II End-Prep Reaction Buffer & Enzyme

20. **Prepare Master Mix for 96 Samples:**
    - Reaction Buffer: 213.59 µL
    - Enzyme: 91.425 µL
    - Mix and distribute into the 96-well plate.

21. **Standardize DNA:**
    - Normalization using qubit data and spreadsheet.
    - Incubate, perform end-repair.

#### Sample Barcoding

22. **Reagents:**
    - Native Barcoding Expansion (EXP NBD 196)
    - NEB Ultra II Ligation Master Mix and Enhancer

23. **Prepare Master Mix:**
    - Nuclease-Free Water: 604.2 µL
    - Ligation Master Mix: 1060 µL
    - Ligation Enhancer: 31.8 µL
    - Mix thoroughly.

#### Adapter Ligation & Final Steps

24. **Reagents:**
    - Adapter Mix II
    - NEB Quick Ligase and Buffer
    - Elution Buffer

25. **Complete Ligation Steps:**
    - Mix Polishes, Spin, Elute, and Store.

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**End of Document**
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