TITLE: HIV-PULSE Wet-lab Protocol
AUTHORS: Laurens Lambrechts, Sofie De Braekeleer, Basiel Cole, Linos Vandekerckhove

[DESCRIPTION]
Protocols.io for HIV-PULSE assay, including an updated protocol from the work published in NAR.

[BEFORE_START]
The HIV-PULSE assay consists of six different PCR steps followed by sequencing on the Minion long read sequencer from Oxford Nanopore Technologies. The main steps are as follows; (i) pre-amplification (to increase sensitivity) and tagging of pre-amplified HIV-1 fragments at each end with a unique UMI, (ii) PCR amplification (split over multiple PCR rounds) will amplify all UMI tagged amplicons making use of the universal synthetic primers attached to each UMI end, (iii) sequencing will be performed on long read sequencer so reads will span the entire HIV-1 fragment with the attached UMI fragments at both ends needed for the bio-informatic workflow (https://github.com/laulambr/longread_umi_hiv). 

[STEPS]
SECTION: Pre-amplification: PCR using PrimeSTAR GXL
2. Prepare the master mix according to this principle (if doing replicates, add DNA to master mix, mix well and then divide per sample to ensure ‘even’ input), beware to adapt the mix according to the required DNA input volume: 
a. x µL DNA input 
b. 33- x µL NFW 
 
 
 
SECTION: Pre-amplification: PCR using PrimeSTAR GXL
1. Calculate the x µL volume of sample needed to have an input of 500 ng of genomic DNA. 

SECTION: Pre-amplification: PCR using PrimeSTAR GXL
3. Use the following PCR cycling conditions on the cycler. The number of cycles can vary depending on the HIV-1 copy number of the input sample, most important requirement is generating enough yield in order to sequence. Some guidelines: 
a. > 2500 total HIV-1 copies/million CD4 T cells: 5 pre-amp cycles 
b. < 2500 total HIV-1 copies/million CD4 T cells: 6 pre-amp cycles  
 
 
SECTION: Pre-amplification: cleanup
4. Cleanup of the PCR product using the original CleanPCR beads (CleanNA) at 1.0x ratio. Thus for 50 µLreaction volume of PCR 1, add 50 µLµL of CleanPCR beads.
SECTION: Pre-amplification: cleanup
5. Make sure the CleanPCR magnetic beads are at room temperature.
SECTION: Pre-amplification: cleanup
6. Prepare a fresh 70% Ethanol solution.
SECTION: Pre-amplification: cleanup
7. Vortex the bead solution for30 sto homogenize the beads.

SECTION: Pre-amplification: cleanup
8. Add50 µLof bead solution to 50 µLof PCR 1 product and mix by flicking. If you have multiple samples, change tip!
SECTION: Pre-amplification: cleanup
9. Incubate for 5 min at room temperature.

SECTION: Pre-amplification: cleanup
10. Spin down in mini centrifuge.
SECTION: Pre-amplification: cleanup
11. Place the sample on a magnet for 2 minor until the supernatant has cleared.

SECTION: Pre-amplification: cleanup
12. Keep sample on magnet, discard supernatant without disturbing the bead pellet. If you have multiple samples, change tip!
SECTION: Pre-amplification: cleanup
13. Wash the beads with fresh 70% Ethanol by adding 400 µL at the opposite side of the beads (if recipient doesn’t hold 400 µL, adapt volume and just make sure that beads are covered in ethanol). Do not resuspend the beads!
SECTION: Pre-amplification: cleanup
14. Incubate for30 s and remove the ethanol.

SECTION: Pre-amplification: cleanup
15. Repeat the washing steps 13 & 14.
SECTION: Pre-amplification: cleanup
16. Remove excess ethanol by using a P20 pipet (optionally you can also spin down the sample and put back on magnet.
SECTION: Pre-amplification: cleanup
17. Let the beads air dry for 30 s or until the pellet loses its shine (but avoid cracking of DNA since this will make resuspending hard).

SECTION: Pre-amplification: cleanup
18. Remove sample from magnet.
SECTION: Pre-amplification: cleanup
19. Elute the purified DNA by adding 35 µL NFW. Mix by flicking the tube.

SECTION: Pre-amplification: cleanup
20. Incubate for 5 minat room temperature.

SECTION: Pre-amplification: cleanup
21. Put sample on magnet for2 minor until the supernatant has cleared.

SECTION: Pre-amplification: cleanup
22. Transfer30 µLof cleaned DNA to new tube.

SECTION: Tagging: PCR
23. Prepare master mix for PCR2 (can be done during pre-amplification PCR). Use the 30 µLof cleaned DNA from previous step as input.  
 
 

SECTION: Tagging: PCR
23.1. Use the inner HIV primers with a UMI tail and not the regular ones. Primers with UMI should be PAGE purified.
SECTION: Tagging: PCR
24. Use the following PCR cycling conditions on the cycler.   
 

SECTION: Tagging: PCR
24.1. Do not change the number of cycles since this would mess up the tagging!
SECTION: Tagging: cleanup
25. Make sure the custom CleanPCR magnetic beads are at room temperature.
SECTION: Tagging: cleanup
26. Prepare a fresh 70% Ethanol solution.
SECTION: Tagging: cleanup
27. Vortex the custom bead solution for 30 sto homogenize the beads.

SECTION: Tagging: cleanup
28. Add 50*ratio µL of bead solution to 50 µL of PCR 2 product and mix by flicking. If you have multiple samples, change tip!

SECTION: Tagging: cleanup
29. Perform clean up as listed in steps 9 to 18.
SECTION: Tagging: cleanup
30. Elute the purified DNA by adding 35 µL Mix by flicking the tube.

SECTION: Tagging: cleanup
31. Incubate for 5 minat room temperature.

SECTION: Tagging: cleanup
32. Put sample on magnet for 2 min or until the supernatant has cleared.

SECTION: Tagging: cleanup
33. Transfer 30 µLof supernatant to new tube.

SECTION: Tagging: cleanup
25.1. Cleanup of the PCR product using the custom CleanPCR beads at a defined ratio for that custom batch.
SECTION: PCR round 1-2-3: amplification of UMI tagged amplicons
34. Prepare master mix for amplification PCR 1 or PCR 2/3 (Mix is different, for PCR1 30 µL input, while in PCR 2-3 10 µL).
a. For PCR 1: use the30 µL of cleaned tagged DNA from tagging step as input.
b. For PCR2-3: use the 10 µL of cleaned DNA from previous step as input. Store the remaining 20 µLat-20 °C.

 
 
 
 
 
 
SECTION: PCR round 1-2-3: amplification of UMI tagged amplicons
35. Use the following PCR cycling conditions on the cycler.  
 
 
SECTION: PCR round 1-2-3: cleanup
36. Make sure the regular CleanPCR magnetic beads are at room temperature.
SECTION: PCR round 1-2-3: cleanup
37. Prepare a fresh 70% Ethanol solution.
SECTION: PCR round 1-2-3: cleanup
38. Vortex the custom bead solution for 30 s to homogenize the beads.

SECTION: PCR round 1-2-3: cleanup
39. Use the original CleanPCR beads (CleanNA) at 1.0x ratio. Thus for 50 µLreaction volume of PCR product, add 50 µLof CleanPCR beads. Mix by flicking. If you have multiple samples, change tip!

SECTION: PCR round 1-2-3: cleanup
40. Perform clean up as listed in steps 9 to 18.
SECTION: PCR round 1-2-3: cleanup
41. Elute the purified DNA by adding 35 µLNFW. Mix by flicking the tube.

SECTION: PCR round 1-2-3: cleanup
42. Incubate for 5 mint room temperature.

SECTION: PCR round 1-2-3: cleanup
43. Put sample on magnet for 2 minor until the supernatant has cleared.

SECTION: PCR round 1-2-3: cleanup
44. Transfer 30 µLof supernatant to new tube.

SECTION: PCR round 4: amplification of UMI tagged amplicons with sample specific primers
45. For the last PCR round, we will tag replicates from the same sample with the same primerset tailed with an index (ID1-8). This will allow later for demultiplexing the replicates per sample based on this index.   
 
 
SECTION: PCR round 4: amplification of UMI tagged amplicons with sample specific primers
46. Prepare master mix for amplification PCR 4. Note that this also uses cresol red and more cleaned material as input to ensure enough yield.
SECTION: PCR round 4: amplification of UMI tagged amplicons with sample specific primers
47. Use the following PCR program. 
 
 
SECTION: PCR round 4: amplification of UMI tagged amplicons with sample specific primers
47.1. PCR program is slightly adapted from other PCR programs to compensate for longer tailed primers and reduce primer/dimer. 
SECTION: PCR round 4: gel
48. After PCR is finished, transfer 5 µL and visualize on a 1% agarose gel (30 min, 120 Volt) with a GeneRuler 1 kb Plus DNA Ladder (SM1333) for reference. When imaging, use the faint settings. 

SECTION: PCR round 4: cleanup, concentration measurement and pooling
49. Make sure the custom CleanPCR magnetic beads are at room temperature.
SECTION: PCR round 4: cleanup, concentration measurement and pooling
50. Prepare a fresh 70% Ethanol solution.
SECTION: PCR round 4: cleanup, concentration measurement and pooling
51. Vortex the custom bead solution for 30 s to homogenize the beads.

SECTION: PCR round 4: cleanup, concentration measurement and pooling
52. Add 45*ratio µL of bead solution to 45 µLof PCR 4 product and mix by flicking. If you have multiple samples, change tip!

SECTION: PCR round 4: cleanup, concentration measurement and pooling
53. Perform clean up as listed in steps 9 to 18.
SECTION: PCR round 4: cleanup, concentration measurement and pooling
54. Elute the purified DNA by adding 18 µL NFW. Mix by flicking the tube.

SECTION: PCR round 4: cleanup, concentration measurement and pooling
55. Incubate for 5 min at room temperature.

SECTION: PCR round 4: cleanup, concentration measurement and pooling
56. Put sample on magnet for2 minor until the supernatant has cleared.

SECTION: PCR round 4: cleanup, concentration measurement and pooling
57. Transfer 15 µLof supernatant to new tube.

SECTION: PCR round 4: cleanup, concentration measurement and pooling
58. Quantify with Picogreen, by taking1 µLof cleaned product as input (performed in duplicate). 

SECTION: PCR round 4: cleanup, concentration measurement and pooling
59. Based on measured DNA concentrations and estimated overall fragment length (~4-6 kb), calculate for each replicate the equimolar sample pooling strategy.
SECTION: PCR round 4: cleanup, concentration measurement and pooling
49.1. Cleanup of the PCR product using the custom CleanPCR beads at a defined ratio for that custom batch.
SECTION: Sequencing
60. For sequencing protocol, see ONT protocols. Use LFB. Each replicate will be run with a different native barcode from the nanopore barcoding kits (NB01-24). If reusing a flowcell, try to avoid including a barcode already used in previous run. 
SECTION: Extra: make CUSTOM SPRI bead solution
62. Step 1: Prepare the custom buffer by mixing: 

 

SECTION: Extra: make CUSTOM SPRI bead solution
63. Step 2: Transfer bead to Custom buffer 

SECTION: Extra: make CUSTOM SPRI bead solution
61. Protocol based on 'SPRI size selection protocol for > 1.5-2 kb DNA fragments' from Oxford Nanopore Technologies.

SECTION: Extra: make CUSTOM SPRI bead solution
63.1. Bring CleanPCR beads (CleanNA) to room temperature. 
SECTION: Extra: make CUSTOM SPRI bead solution
63.2. Vortex for30 s to resuspend beads properly. 

SECTION: Extra: make CUSTOM SPRI bead solution
63.3. Transfer beads into two 1.5 mL tubes so each contains 1 mL.

SECTION: Extra: make CUSTOM SPRI bead solution
63.4. Place the tubes on the magnet, wait until the solution is clear and discard the supernatant. 

SECTION: Extra: make CUSTOM SPRI bead solution
63.5. Remove the tubes from the magnet, wash with 1 mLof NFW by resuspending the pellet. 

SECTION: Extra: make CUSTOM SPRI bead solution
63.6. Return the tubes to the magnet, allow beads to pellet and pipette of the supernanant.
SECTION: Extra: make CUSTOM SPRI bead solution
63.7. Repeat this NFW wash once more.
SECTION: Extra: make CUSTOM SPRI bead solution
63.8. Spin down and place tubes back on magnet. Pipette off any residual water. 
SECTION: Extra: make CUSTOM SPRI bead solution
63.9. Pool the two bead pellets together by resuspending them in200 µLof Custom buffer.

SECTION: Extra: make CUSTOM SPRI bead solution
63.10. Transfer the beads into the remaining Custom buffer. 

SECTION: Extra: make CUSTOM SPRI bead solution
64. Step 3: Test different ratios of new batch of Custom buffer on DNA ladder to determine perfect ratio.
We generally test a range of different ratios including 0.8x, 0.9x, 1.0x, 1.1x and 1.2x conditions.

 
  

SECTION: Extra: make CUSTOM SPRI bead solution
64.1. For each ratio to be tested, take 3 µLof GeneRuler 1 kb DNA Ladder (SM0311), add to 17 µLof TE.    

SECTION: Extra: make CUSTOM SPRI bead solution
64.4. Put on magnet for two minutes or until supernatant has cleared. 
SECTION: Extra: make CUSTOM SPRI bead solution
64.3. Incubate for5 minat room temperature. 

SECTION: Extra: make CUSTOM SPRI bead solution
64.2. Add to this the required volume of custom buffer (for 1.0x add for instance 20 µL).

SECTION: Extra: make CUSTOM SPRI bead solution
64.5. Discard supernatant. 
SECTION: Extra: make CUSTOM SPRI bead solution
64.10. Take from magnet and resuspend, incubate for 5 min

SECTION: Extra: make CUSTOM SPRI bead solution
64.9. Add30 µLNFW buffer. 

SECTION: Extra: make CUSTOM SPRI bead solution
64.8. Remove residual ethanol.
SECTION: Extra: make CUSTOM SPRI bead solution
64.7. Repeat ethanol step once more.
SECTION: Extra: make CUSTOM SPRI bead solution
64.6. Wash by adding 200 µL70% ethanol, leave 30 s and remove.

SECTION: Extra: make CUSTOM SPRI bead solution
64.12. Remove 30 µL cleaned ladder and put on visualize on a 1% agarose gel. 


SECTION: Extra: make CUSTOM SPRI bead solution
64.11. Put on magnet for 2 minuntil supernatant has cleared. 

SECTION: Pre-amplification: PCR using PrimeSTAR GXL
1.1. More important than the 500 ngis the actual HIV-1 input copies. Minimum 30 copies based on total HIV-1 measurements is advised to obtain at least a single genome.