HIV-PULSE Wet-lab Protocol

Calculate the x µL volume of sample needed to have an input of 500ng of genomic DNA.

More important than the 500ngis the actual HIV-1 input copies. Minimum 30 copies based on total HIV-1 measurements is advised to obtain at least a single genome.

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 

A	B	C	D
	Stock conc.	Final conc.	Quantity per rx (µL)
DNA			1
5X PrimeSTAR GXL Buffer	5X	1X	10
dNTP Mixture (PS GXL)	2,5 mM	200 µM each	4
Pinzone First PCR F	10 µM	0,2 µM	1
Pinzone First PCR R	10 µM	0,2 µM	1
PrimeSTAR GXL DNA Polymerase	1.25 U/µL	1,25 U / 50 µl	1
Nuclease free water	-	-	32
Total			50

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 

A	B	C
STEP	TEMP	TIME
Initial Denaturation	98°C	2 minutes
x Cycles	98°C	10 seconds
	65°C (Pinzone)	15 seconds
	68°C	10 minutes
Final Extension	68°C	10 minutes
Hold	4-10°C

Cleanup of the PCR product using the original CleanPCR beads (CleanNA) at 1.0x ratio. Thus for 50µLreaction volume of PCR 1, add 50µLµL of CleanPCR beads.

Make sure the CleanPCR magnetic beads are at room temperature.

Prepare a fresh 70% Ethanol solution.

Vortex the bead solution for0h 0m 30sto homogenize the beads.

Add50µLof bead solution to 50µLof PCR 1 product and mix by flicking. If you have multiple samples, change tip!

Incubate for 0h 5m 0s at room temperature.

Spin down in mini centrifuge.

Place the sample on a magnet for 0h 2m 0sor until the supernatant has cleared.

Keep sample on magnet, discard supernatant without disturbing the bead pellet. If you have multiple samples, change tip!

Wash the beads with fresh 70% Ethanol by adding 400µL at the opposite side of the beads (if recipient doesn’t hold 400µL, adapt volume and just make sure that beads are covered in ethanol). Do not resuspend the beads!

Incubate for0h 0m 30s and remove the ethanol.

Repeat the washing steps 13 & 14.

Remove excess ethanol by using a P20 pipet (optionally you can also spin down the sample and put back on magnet.

Let the beads air dry for 0h 0m 30s or until the pellet loses its shine (but avoid cracking of DNA since this will make resuspending hard).

Remove sample from magnet.

Elute the purified DNA by adding 35µL NFW. Mix by flicking the tube.

Incubate for 0h 5m 0sat room temperature.

Put sample on magnet for0h 2m 0sor until the supernatant has cleared.

Transfer30µLof cleaned DNA to new tube.

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

A	B	C	D
	Stock conc.	Final conc.	Quantity per rx (µL)
cleaned PCR 1 supernatant			30
5X LongAmp Taq Reaction Buffer	5x	1x	10
dNTP mix	10 mM	100 µM	1.5
Inner UMI HIV PCR Fwd	10 µM	500 nM	2.5
Inner UMI HIV PCR Rev	10 µM	500 nM	2.5
LongAmp Taq DNA Polymerase	5U/µL	0.5U	2
Nuclease free water	-	-	1.5
			50

Use the inner HIV primers with a UMI tail and not the regular ones. Primers with UMI should be PAGE purified.

Use the following PCR cycling conditions on the cycler.  

A	B	C
STEP	TEMP	TIME
Initial Denaturation	94°C	1m 15 seconds
2 Cycles	94°C	30 seconds
	58 °C	30 seconds
	65°C	10 minutes
Final Extension	65°C	10 minutes
Hold	4-10°C

Do not change the number of cycles since this would mess up the tagging!

Make sure the custom CleanPCR magnetic beads are at room temperature.

Cleanup of the PCR product using the custom CleanPCR beads at a defined ratio for that custom batch.

Prepare a fresh 70% Ethanol solution.

Vortex the custom bead solution for 0h 0m 30sto homogenize the beads.

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

Perform clean up as listed in steps 9 to 18.

Elute the purified DNA by adding 35µL Mix by flicking the tube.

Incubate for 0h 5m 0sat room temperature.

Put sample on magnet for 0h 2m 0s or until the supernatant has cleared.

Transfer 30µLof supernatant to new tube.

Prepare master mix for amplification PCR 1 or PCR 2/3 (Mix is different, for PCR1 30µL input, while in PCR 2-3 10µL).

a. For PCR 1: use the30µL of cleaned tagged DNA from tagging step as input.

b. For PCR2-3: use the 10µL of cleaned DNA from previous step as input. Store the remaining 20µLat-20°C.

A	B	C	D
Component	Stock conc.	Final conc.	Quantity (µL)
Eluted UMI tagged DNA			30
5X LongAmp Taq Reaction Buffer	10x	1x	10
dNTP mix	10 mM	200 µM	1.5
Forward_PCR_ONT	10 µM	500 nM	2.5
Reverse_PCR_ONT	10 µM	500 nM	2.5
LongAmp Taq DNA Polymerase	5U/µL	1.25U	2
Nuclease free water	-	-	1.5
Total			50

PCR mix for PCR 1 

A	B	C	D
Component	Stock conc.	Final conc.	Quantity (µL)
Eluted UMI tagged DNA			30
5X LongAmp Taq Reaction Buffer	10x	1x	10
dNTP mix	10 mM	200 µM	1.5
Forward_PCR_ONT	10 µM	500 nM	2.5
Reverse_PCR_ONT	10 µM	500 nM	2.5
LongAmp Taq DNA Polymerase	5U/µL	1.25U	2
Nuclease free water	-	-	1.5
Total			50

PCR mix for PCR 2-3 

Use the following PCR cycling conditions on the cycler. 

A	B	C
STEP	TEMP	TIME
Initial Denaturation	94°C	1m 15 seconds
10 Cycles	94°C	30 seconds
	60 °C	30 seconds
	65°C	10 minutes
Final Extension	65°C	10 minutes
Hold	4-10°C

Make sure the regular CleanPCR magnetic beads are at room temperature.

Prepare a fresh 70% Ethanol solution.

Vortex the custom bead solution for 0h 0m 30s to homogenize the beads.

Use the original CleanPCR beads (CleanNA) at 1.0x ratio. Thus for 50µLreaction volume of PCR product, add 50µLof CleanPCR beads. Mix by flicking. If you have multiple samples, change tip!

Perform clean up as listed in steps 9 to 18.

Elute the purified DNA by adding 35µLNFW. Mix by flicking the tube.

Incubate for 0h 5m 0st room temperature.

Put sample on magnet for 0h 2m 0sor until the supernatant has cleared.

Transfer 30µLof supernatant to new tube.

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.  

A	B	C	D
Component	Stock conc.	Final conc.	Quantity (µL)
Eluted UMI tagged DNA			20
5X LongAmp Taq Reaction Buffer	10x	1x	10
dNTP mix	10 mM	200 µM	1.5
Forward_PCR_ONT	10 µM	500 nM	2.5
Reverse_PCR_ONT	10 µM	500 nM	2.5
LongAmp Taq DNA Polymerase	5U/µL	1.25U	2
Cresol red	10x	1x	5
Nuclease free water	-	-	6.5
Total			50

PCR mix for PCR 4

Prepare master mix for amplification PCR 4. Note that this also uses cresol red and more cleaned material as input to ensure enough yield.

Use the following PCR program.

A	B	C
STEP	TEMP	TIME
Initial Denaturation	94°C	1m 15 seconds
10 Cycles	94°C	30 seconds
	61°C	30 seconds
	65°C	10 minutes
Final Extension	65°C	10 minutes
Hold	4-10°C

PCR program is slightly adapted from other PCR programs to compensate for longer tailed primers and reduce primer/dimer. 

After PCR is finished, transfer 5µL and visualize on a 1% agarose gel (0h 30m 0s, 120 Volt) with a GeneRuler 1 kb Plus DNA Ladder (SM1333) for reference. When imaging, use the faint settings. 

Make sure the custom CleanPCR magnetic beads are at room temperature.

Cleanup of the PCR product using the custom CleanPCR beads at a defined ratio for that custom batch.

Prepare a fresh 70% Ethanol solution.

Vortex the custom bead solution for 0h 0m 30s to homogenize the beads.

Add 45*ratio µL of bead solution to 45µLof PCR 4 product and mix by flicking. If you have multiple samples, change tip!

Perform clean up as listed in steps 9 to 18.

Elute the purified DNA by adding 18µL NFW. Mix by flicking the tube.

Incubate for 0h 5m 0s at room temperature.

Put sample on magnet for0h 2m 0sor until the supernatant has cleared.

Transfer 15µLof supernatant to new tube.

Quantify with Picogreen, by taking1µLof cleaned product as input (performed in duplicate).

Based on measured DNA concentrations and estimated overall fragment length (~4-6 kb), calculate for each replicate the equimolar sample pooling strategy.

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. 

Protocol based on 'SPRI size selection protocol for > 1.5-2 kb DNA fragments' from Oxford Nanopore Technologies.

Step 1: Prepare the custom buffer by mixing: 

A	B	C
Final	Stock	Input (µL)
10 mM Tris-HCl	1 M	20
1 mM EDTA pH 8	0.5 M	4
1.6 M NaCl	5 M	640
11% PEG 8000	50% (w/v)	440
Nuclease free water	-	888
Total		1992

Step 2: Transfer bead to Custom buffer 

Bring CleanPCR beads (CleanNA) to room temperature.

Transfer the beads into the remaining Custom buffer. 

Vortex for0h 0m 30s to resuspend beads properly.

Transfer beads into two 1.5mL tubes so each contains 1mL.

Place the tubes on the magnet, wait until the solution is clear and discard the supernatant.

Remove the tubes from the magnet, wash with 1mLof NFW by resuspending the pellet.

Return the tubes to the magnet, allow beads to pellet and pipette of the supernanant.

Repeat this NFW wash once more.

Spin down and place tubes back on magnet. Pipette off any residual water.

Pool the two bead pellets together by resuspending them in200µLof Custom buffer.

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.

A
new 0.8
new 0.9
new 1.0
new 1.1
new 1.2

 

For each ratio to be tested, take 3µLof GeneRuler 1 kb DNA Ladder (SM0311), add to 17µLof TE.

Take from magnet and resuspend, incubate for 0h 5m 0s

Put on magnet for 0h 2m 0suntil supernatant has cleared.

Remove 30µL cleaned ladder and put on visualize on a 1% agarose gel.

Add to this the required volume of custom buffer (for 1.0x add for instance 20µL).

Incubate for0h 5m 0sat room temperature.

Put on magnet for two minutes or until supernatant has cleared.

Discard supernatant.

Wash by adding 200µL70% ethanol, leave 0h 0m 30s and remove.

Repeat ethanol step once more.

Remove residual ethanol.

Add30µLNFW buffer.