Aug 02, 2024
Version 2
HIV-PULSE Wet-lab Protocol V.2
- Laurens Lambrechts1,
- Sofie De Braekeleer1,
- Basiel Cole1,
- Linos Vandekerckhove1
- 1UGent
Protocol Citation: Laurens Lambrechts, Sofie De Braekeleer, Basiel Cole, Linos Vandekerckhove 2024. HIV-PULSE Wet-lab Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rby4g1b/v2Version created by Laurens Lambrechts
Manuscript citation:
Laurens Lambrechts, Noah Bonine, Rita Verstraeten, Marion Pardons, Ytse Noppe, Sofie Rutsaert, Filip Van Nieuwerburgh, Wim Van Criekinge, Basiel Cole, Linos Vandekerckhove, HIV-PULSE: a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization, Nucleic Acids Research, Volume 51, Issue 20, 10 November 2023, Page e102, https://doi.org/10.1093/nar/gkad790
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: Working
We use this protocol and it's working
Created: August 02, 2024
Last Modified: August 02, 2024
Protocol Integer ID: 104466
Keywords: nanopore, hiv, umi, hiv, pulse wet, updated protocol, pulse, assay, protocol
Abstract
Protocols.io for HIV-PULSE assay, including an updated protocol from the work published in NAR.
Materials
Qubit dsDNA HS Assay Kit (Q32854)
Quant-iT PicoGreen dsDNA Assay Kit (P7589)
CleanPCR beads (CPCR-0050)
Custom size select CleanPCR beads
LongAmp Hot Start Taq DNA Polymerase (M0534L)
dNTPs (Promega, C1141)
PrimeSTAR GXL DNA Polymerase (R050A)
GeneRuler 1 kb Plus DNA Ladder, ready to use (SM1333)
GeneRuler 1 kb DNA Ladder (SM0311)
Native Barcoding Kit 24 V14 kit (ONT, SQK-NBD114-24)
MinION R10.4.1 flow cells (ONT, FLO-MIN114)
Primers (IDT)
| A | B | C | D | |
| PCR-step | Assay | Name | Sequence (5’ to 3’) | |
| Pre-amplification | Pinzone outer | Pinzone First PCR Fwd | CCTCAATAAAGCTTGCCTTGAGTGC | |
| Pinzone First PCR Rev | CCTAGTTAGCCAGAGAGCTCCCAG | |||
| FLIPS/Lee outer | BLOuterF; U5-623F | AAATCTCTAGCAGTGGCGCCCGAACAG | ||
| BLOuterR; U5-601R | TGAGGGATCTCTAGTTACCAGAGTC | |||
| Tagging (PAGE purified) | Pinzone inner | UMI_Pinzone_F2 | CAAGCAGAAGACGGCATACGAGATNNNYRNNNYRNNNYRNNNAAGTAGTGTGTGCCCGTCTGTTGTGTGAC | |
| UMI_Pinzone_R2 | AATGATACGGCGACCACCGAGATCNNNYRNNNYRNNNYRNNNGGAAAGTCCCCAGCGGAAAGTCCCTTGTAG | |||
| Lee inner | UMI_Lee_F2 | CAAGCAGAAGACGGCATACGAGATNNNYRNNNYRNNNYRNNNGCGCCCGAACAGGGACYTGAAARCGAAAG | ||
| UMI_Lee_R2 | AATGATACGGCGACCACCGAGATCNNNYRNNNYRNNNYRNNNGCACTCAAGGCAAGCTTTATTGAGGCTTA | |||
| Amplification (PCR1 to PCR3) | PCR1 to 3 | ncec_pcr_fw_v7 | CAAGCAGAAGACGGCATACGAGAT | |
| ncec_pcr_rv_v7 | AATGATACGGCGACCACCGAGATC | |||
| Patient barcoding (PCR4) | ID1 | ONT_fw1 | ACGAGACTGATTCAAGCAGAAGACGGCATACGAGAT | |
| ONT_UD1_R | ACGAGACTGATTAATGATACGGCGACCACCGAGATC | |||
| ID2 | ONT_fw2 | GCTGTACGGATTCAAGCAGAAGACGGCATACGAGAT | ||
| ONT_UD2_R | GCTGTACGGATTAATGATACGGCGACCACCGAGATC | |||
| ID3 | ONT_fw3 | ATCACCAGGTGTCAAGCAGAAGACGGCATACGAGAT | ||
| ONT_UD3_R | ATCACCAGGTGTAATGATACGGCGACCACCGAGATC | |||
| ID4 | ONT_fw4 | TGGTCAACGATACAAGCAGAAGACGGCATACGAGAT | ||
| ONT_UD4_R | TGGTCAACGATAAATGATACGGCGACCACCGAGATC | |||
| ID5 | ONT_fw5 | ATCGCACAGTAACAAGCAGAAGACGGCATACGAGAT | ||
| ONT_UD5_R | ATCGCACAGTAAAATGATACGGCGACCACCGAGATC | |||
| ID6 | ONT_fw6 | GTCGTGTAGCCTCAAGCAGAAGACGGCATACGAGAT | ||
| ONT_UD6_R | GTCGTGTAGCCTAATGATACGGCGACCACCGAGATC | |||
| ID7 | ONT_fw7 | AGCGGAGGTTAGCAAGCAGAAGACGGCATACGAGAT | ||
| ONT_UD7_R | AGCGGAGGTTAGAATGATACGGCGACCACCGAGATC | |||
| ID8 | ONT_fw8 | ATCCTTTGGTTCCAAGCAGAAGACGGCATACGAGAT | ||
| ONT_UD8_R | ATCCTTTGGTTCAATGATACGGCGACCACCGAGATC |
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).
Pre-amplification: PCR using PrimeSTAR GXL
Calculate the x µL volume of sample needed to have an input of 500 ng of genomic DNA.
More important than the 500 ng is 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
| 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 |
Pre-amplification: cleanup
Cleanup of the PCR product using the original CleanPCR beads (CleanNA) at 1.0x ratio. Thus for 50 µL reaction volume of PCR 1, add 50 µL µL of CleanPCR beads.
Make sure the CleanPCR magnetic beads are at room temperature.
Prepare a fresh 70% Ethanol solution.
Vortex the bead solution for00:00:30 to homogenize the beads.
30s
Add50 µL of bead solution to 50 µL of PCR 1 product and mix by flicking. If you have multiple samples, change tip!
Incubate for 00:05:00 at room temperature.
5m
Spin down in mini centrifuge.
Place the sample on a magnet for 00:02:00 or until the supernatant has cleared.
2m
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 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!
Incubate for00:00:30 and remove the ethanol.
30s
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 00:00:30 or until the pellet loses its shine (but avoid cracking of DNA since this will make resuspending hard).
30s
Remove sample from magnet.
Elute the purified DNA by adding 35 µL NFW. Mix by flicking the tube.
Incubate for 00:05:00 at room temperature.
5m
Put sample on magnet for00:02:00 or until the supernatant has cleared.
2m
Transfer30 µL of cleaned DNA to new tube.
Tagging: PCR
Prepare master mix for PCR2 (can be done during pre-amplification PCR). Use the 30 µL of cleaned DNA from previous step as input.
| 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 |
34m
Do not change the number of cycles since this would mess up the tagging!
Tagging: cleanup
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 00:00:30 to homogenize the beads.
30s
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!
Perform clean up as listed in steps 9 to 18.
Elute the purified DNA by adding 35 µL Mix by flicking the tube.
Incubate for 00:05:00 at room temperature.
5m
Put sample on magnet for 00:02:00 or until the supernatant has cleared.
2m
Transfer 30 µL of supernatant to new tube.
PCR round 1-2-3: amplification of UMI tagged amplicons
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 µL at-20 °C .
| 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
| 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 |
PCR round 1-2-3: cleanup
Make sure the regular CleanPCR magnetic beads are at room temperature.
Prepare a fresh 70% Ethanol solution.
Vortex the custom bead solution for 00:00:30 to homogenize the beads.
30s
Use the original CleanPCR beads (CleanNA) at 1.0x ratio. Thus for 50 µL reaction volume of PCR product, add 50 µL of 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 35 µL NFW. Mix by flicking the tube.
Incubate for 00:05:00 t room temperature.
5m
Put sample on magnet for 00:02:00 or until the supernatant has cleared.
2m
Transfer 30 µL of supernatant to new tube.
PCR round 4: amplification of UMI tagged amplicons with sample specific primers
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.
| 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.
PCR round 4: gel
After PCR is finished, transfer 5 µL and visualize on a 1% agarose gel (00:30:00 , 120 Volt) with a GeneRuler 1 kb Plus DNA Ladder (SM1333) for reference. When imaging, use the faint settings.
30m
PCR round 4: cleanup, concentration measurement and pooling
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 00:00:30 to homogenize the beads.
30s
Add 45*ratio µL of bead solution to 45 µL of 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 18 µL NFW. Mix by flicking the tube.
Incubate for 00:05:00 at room temperature.
5m
Put sample on magnet for00:02:00 or until the supernatant has cleared.
2m
Transfer 15 µL of supernatant to new tube.
Quantify with Picogreen, by taking1 µL of 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.
Sequencing
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.
Extra: make CUSTOM SPRI bead solution
30s
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:
| 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.
Vortex for00:00:30 to resuspend beads properly.
30s
Transfer beads into two 1.5 mL tubes so each contains 1 mL .
Place the tubes on the magnet, wait until the solution is clear and discard the supernatant.
Remove the tubes from the magnet, wash with 1 mL of 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 in200 µL of Custom buffer.
Transfer the beads into the remaining 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.
| new 0.8 | |
| new 0.9 | |
| new 1.0 | |
| new 1.1 | |
| new 1.2 |
For each ratio to be tested, take 3 µL of GeneRuler 1 kb DNA Ladder (SM0311), add to 17 µL of TE.
Add to this the required volume of custom buffer (for 1.0x add for instance 20 µL ).
Incubate for00:05:00 at room temperature.
5m
Put on magnet for two minutes or until supernatant has cleared.
Discard supernatant.
Wash by adding 200 µL 70% ethanol, leave 00:00:30 and remove.
30s
Repeat ethanol step once more.
Remove residual ethanol.
Add30 µL NFW buffer.
Take from magnet and resuspend, incubate for 00:05:00
5m
Put on magnet for 00:02:00 until supernatant has cleared.
2m
Remove 30 µL cleaned ladder and put on visualize on a 1% agarose gel.