TITLE: CMV Resistance testing (UL54 and UL97)
AUTHORS: Fernando Lazaro

[DESCRIPTION]
This protocol is a procedure for the study of antiviral resistance in Cytomegalovirus by NGS techniques.
The primers have been designed using https://primalscheme.com/ with the intention of covering the most relevant regions of the UL54 and UL97 genes.

[BEFORE_START]
Take into account that the quality of the results is greatly affected by the time from sample extraction to DNA amplification.
The protocol may fail if the protocol is performed from refrigerated samples or DNA.

[STEPS]
SECTION: Prepare Reagents
1. Q5‱ High-Fidelity DNA Polymerase (New England)
Agarose gel 1%
Ethanol 70%
Mag-Bind‱ TotalPure NGS (omega)
Elution Buffer
SECTION: DNA extraction
3. Perform DNA extraction with your method of choice. Preferably from a plasma sample collected on the same day.Perform DNA extraction with your method of choice. Preferably from a plasma sample collected on the same day.

The quality of the results is greatly affected by the time from sample extraction to DNA amplification.
The protocol may fail if the protocol is performed from frozen samples or DNA.
SECTION: DNA amplification
4.   For each pool and sample, mix the following reagents (two reactions per sample):

 
  
  Reagent
  
  
  Volume
  / sample (µl) 
  
  
  
  Primer
  Pool
  
  
  2
  
  
  
  Q5‱ Polymerase
  
  
  0,25
  
  
  
  Q5 Buffer 
  
  
  5
  
  
  
  H20 
  
  
  5
  
  
  
  dNTP 
  
  
  0.5
  
  
  
  Sample DNA 
  
  
  12,5
  
 
 
Set up the PCR with the following program

 
 
  
  Temperature
  
  
  Time
  
  
  
  98°C 
  
  
  30’’ 
  
  
  
  98°C 
  
  
  10’’ 
  
  
  
  65°C 
  
  
  3’ 
  
  
  
  98°C 
  
  
  10’’
  
  
  
  65°C 
  
  
  30’’
  
  
  
  72°C 
  
  
  2’ 
  
  
  
  72°C 
  
  
  2’ 
  
  
  
  4°C 
  
  
  ∞ 
  
 
 
Confirm amplification of aproximately 300bp fragments by 1% agarose gel.

SECTION: Set up primer pools (UL97 and UL54)
2. UL97 Primers
 
  
 
  
  UL97_4_LEFT
  
  
  TGCGCGCGGAAAGTCAG
  
  
  UL97_4_RIGHT
  
  
  CGGCATAACAGATCTTGTGGC
  
  
  
  UL97_5_LEFT
  
  
  CTCTGCGAGCTCTCTATCTCCT
  
  
  UL97_5_RIGHT
  
  
  AGCAGACAGCAGCCCGT
  
  
  
  UL97_6_LEFT
  
  
  TGGCGAGCAACAGCAGC
  
  
  UL97_6_RIGHT
  
  
  GCGCGCATGATCTCGCT
  
  
  
  UL97_7_LEFT
  
  
  TGCCACTTTGACATTACACCCA
  
  
  UL97_7_RIGHT
  
  
  TCCGACATGCAATAACGCCG
  
  
  
  UL97_8_LEFT
  
  
  TTTCCGACCCATGCCGCT
  
  
  UL97_8_RIGHT
  
  
  ATGCTCGCCCAGGAGACAG
  
  
  
  UL97_9_LEFT
  
  
  CATGGGTACGGAGGCGTTG
  
  
  UL97_9_RIGHT
  
  
  GGCCAACAGACGCTCCA
  
 
 
UL54 Primers
 
 
  
  UL54_1_LEFT
  
  
  TGCAAAAACTTGTCCTTGCGC
  
  
  UL54_1_RIGHT
  
  
  ATTCTGTAACCACCGGCGTG
  
  
  
  UL54_2_LEFT
  
  
  GTAGTTGCACACGGCCGAC
  
  
  UL54_2_RIGHT
  
  
  CGTCAATCTAACCTGCCGCA
  
  
  
  UL54_3_LEFT
  
  
  CGTAAAAGACCCGATCCCCG
  
  
  UL54_3_RIGHT
  
  
  TCTCGCTGCTCTTTGAGGATC
  
  
  
  UL54_4_LEFT
  
  
  CTTCATCGAGTGAGAGGCGC
  
  
  UL54_4_RIGHT
  
  
  AGGCTTTGGTGGCGCGT
  
  
  
  UL54_5_LEFT
  
  
  GCTTGACGGGCTCCACAAAA
  
  
  UL54_5_RIGHT
  
  
  GGCGCGGTTCATCAAAGACA
  
  
  
  UL54_6_LEFT
  
  
  TCCCGCGTTCCCACTACATA
  
  
  UL54_6_RIGHT
  
  
  CAACAAGTGGGTTTCGCAGC
  
  
  
  UL54_7_LEFT
  
  
  ATACGGCGCACAGGGTCTT
  
  
  UL54_7_RIGHT
  
  
  GTGTTTGAGCCCGAGGTGG
  
  
  
  UL54_8_LEFT
  
  
  AGTAGCAGAGGTTGTGAGCCA
  
  
  UL54_8_RIGHT
  
  
  GGTTCTGTGGCGGCTATGTT
  
  
  
  UL54_9_LEFT
  
  
  AAACGCCGTCCTGACTCGA
  
  
  UL54_9_RIGHT (V2)
  
  
  CTTGCAATCTGCGCCGTC
  
  
  
  UL54_10_LEFT
  
  
  GGATCTGCTGTCCGTCAAAGA
  
  
  UL54_10_RIGHT
  
  
  ATATTGCGGGTTCGGTGGTT
  
  
  
  UL54_11_LEFT
  
  
  TGTTGAGCTTATAGTTGGGCGA
  
  
  UL54_11_RIGHT
  
  
  CGGCCTTTGTGACCGGTTAC
  
  
  
  UL54_12_LEFT
  
  
  CCTTATACAGGTACTCGAGGCG
  
  
  UL54_12_RIGHT
  
  
  GTGCTACGAGACGGGAGGA
  
  
  
  UL54_13_LEFT
  
  
  AAGTGCAGCCCCGACCAT
  
  
  UL54_13_RIGHT
  
  
  GGATCACCACGTTCGGCTG
  
  
  
  UL54_14_LEFT
  
  
  CCTCGATATCACAAGTCGACGC
  
  
  UL54_14_RIGHT
  
  
  GGCGAACTAGTGCCCGAAC
  
  
  
  UL54_15_LEFT
  
  
  CCGTACCCGTAGATGGAGGT
  
  
  UL54_15_RIGHT
  
  
  GGGACCTATTCGTTTTCACACCTA
  
  
  
  UL54_16_LEFT
  
  
  ACGATAGCGCGGCGACA
  
  
  UL54_16_RIGHT
  
  
  CGGCGTCAGCGTTTGCA
  
 

SECTION: Set up primer pools (UL97 and UL54)
2.1. Pool 1 UL97 (odd primers LEFT and RIGHT)
Pool 2 UL97(even primers LEFT and RIGHT)
Use 10 µL for each primer at 10 micromolar (µM) 

SECTION: Set up primer pools (UL97 and UL54)
2.2. Pool 1 UL54 (odd primers LEFT and RIGHT)
Pool 2 UL54 (even primers LEFT and RIGHT)
Use 10 µL for each primer 10 micromolar (µM)  except:
UL54_14_LEFT/RIGHT 14: 2,5 µl 
UL54_5_LEFT/RIGHT: 5 µl 
UL54_13_LEFT/RIGHT: 5 µl 
UL54_15_LEFT/RIGHT: 20 µl 

SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
5. Add 40 uL of beads to 20 uL of amplicon. Mix.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
6. Incubate the mixture for 5 minutes at room temperature.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
7. Place the tube in the magnet until the solution becomes clear.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
8. Gently remove the supernatant by pipette.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
9. Add 180 uL of 70% ethanol. Mix without breaking the pellet.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
10. Gently discard the ethanol by pipette.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
11. Incubate for 2 minutes at room temperature.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
12. Remove any remaining ethanol. Note: The pellet must not be allowed to dry excessively. If it does occur, the pellet will appear black and cracked.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
13. Remove the tube from the magnet.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
14. Add 25 uL of EB. Mix. Note: do not break the pellet, just peel it away from the wall of the tube.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
15. Incubate for 2 minutes at room temperature.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
16. Place the tube back in the magnet until the solution clears.
SECTION: Product cleaning with Mag-Bind TotalPure NGS (omega)
17. Transfer 20 uL of the supernatant to a new tube.
SECTION: NGS sequencing
18. Sequence the amplicons using the sequencer of choice according to the manufacturer's instructions.
SECTION: Antiviral resistance
19. Generate consensus sequence from the amplicons using the bioinformatics procedure of choice. 
Take into account that in cases of previous exposure to antivirals or prolonged treatment, minority variants may appear.
The website http://cmv-resistance.ucl.ac.uk/herpesdrg/ is helpful for the study of resistance mutations.