Sample preparation and RNA extraction on NucliSENS easyMAG

Retrieve plasma samples from -80°C freezer and leave to equilibrate to room temperature prior to processing.

Vortex magnetic silica briefly and add 50µL to numbered NucliSENS easyMAG consumables.

Using a BioHit multichannel pipette set at P3, mix sample solution with magnetic silica by pipetting up and down, avoiding bubbles. Remove excess bubbles if necessary.

Load NucliSENS easyMAG consumables on to the NucliSENS easyMAG instrument and scan barcodes on consumables.

Program NucliSENS easyMAG instrument and select 25µL elution volume.

Start extraction run.

After extraction is complete, transfer eluent into appropriately labelled1.5mL microcentrifuge tubes.

Discard NucliSENSeasyMAG consumables appropriately as biohazard waste.

Proceed to perform PCR amplification or store RNA at -80°C until use.

Transfer 500µLof plasma into well labeled 1.5mL Sarstedt screw-cap tubes/ eppendorf tubes and place in a pre-cooled microcentrifuge at 4°C.

Mark the top of each tube on the outside and spin at 23000x g,0h 0m 0s for 1h 0m 0sat 4°C.

After spinning, remove 300µL of supernatant and briefly vortex the ~200µL pellet.

Proceed to extraction room

Pipette mix the 200µL pellet and transfer to400µL of lysis buffer.

Vortex briefly and incubate at room temperature for 0h 20m 0s.

While the sample is lysing, label disposable NucliSENS easyMAG consumables according to sample list.

After incubation, transfer lysate (~600µL) into respective wells of NucliSENS easyMAG consumables.

Preparation of one-step reverse transcription and second-round PCR master mix

Proceed to PCR master mix room (i.e. clean room).

All reagents except the enzymes (Superscript IV and Platinum Taq DNA Polymerase) must be thawed and vortexed for 0h 0m 5s to 0h 0m 10s

Label reverse transcription and second-round PCR tubes 0.2mL with sample numbers, accordingly.

Label two 1.5mL microcentrifuge tubes as follows; Tube 1: for RT one-step PCR master mix, and Tube 2: for second-round PCR master mix.

Prepare RT-PCR master mix as shown in Table 1, using Superscript IV one-step PCR reagents.

A	B	C
Reagents	Volume per reaction (µl)	Concentration per reaction
2X Reaction RT-PCR master mix	12.5	1X
Nuclease-free Water	2.25	-
PANA2AF (5µM)	2.5	0.5µM
PANA3AR (5µM)	2.5	0.5µM
SSIV/ Platinum SuperFi DNA polymerase (2X)	0.25	0.02X
Total volume	20	-

Table 1. One-step reverse transcription PCR master mix.

A	B	C
Primer name (Direction)	Primer sequence	HXB2
PANA2AF (Forward)	GAGGCAATGAGCCAARCAAACA	1882 - 1903
PANA3AR (Reverse)	TTCCAGGGCTCTAGKTTAGG	5846 - 5865

Table 2. Details of primers used in one-step reverse transcription PCR master mix.

Prepare master mix for the second-round PCR as shown in Table 3, using Platinum Taq PCR reagents.

A	B	C
Second-round PCR Mastermix
Reagent	Volume per reaction (µl)	Concentration per reaction
Nuclease-free Water	18.4	-
10X PCR Buffer	2.5	1X
MgCl2 (50mM)	1.0	2mM
dNTP (10mM)	0.5	0.2mM
Pro1 (5µM)	0.25	0.05µM
5066R (5µM)	0.25	0.05µM
Platinum Taq DNA Polymerase	0.1	-
Total volume	23	-

Table 3. Second-round PCR master mix.

Details of primers for second-round PCR master mix are shown in Table 4.

A	B	C
Primer name (Direction)	Primer sequences	HXB2
Pro1 (Forward)	TAGAGCCAACAGCCCCACCA	2147 - 2166
5066R (Reverse)	ATCATCACCTGCCATCTGTTTTCCAT	5041 - 5066

Table 4. Details of primers used in second-round PCR.

Mix one-step RT-PCR master mix by pipetting up and down (or by gently tapping the tube), and transfer 20µLto the respective 0.2µL PCR tubes.

Mix second-round PCR master mix by pipetting up and down (or by gently tapping the tube), and transfer 23µLto the respective 0.2µL PCR tubes.

Take aliquoted one-step RT-PCR master mix into the extraction laboratory (or dead air space cabinet in general laboratory).

Adding RNA to one-step RT-PCR master mix.

Proceed to extraction laboratory, or dead air space cabinet.

Thaw RNA, gently tap tubes, and briefly spin.

Add 5µL RNA to respective tubes containing master mix.

Proceed to the PCR amplification room.

RT-PCR amplification process.

Gently tap PCR tubes to mix and centrifuge briefly.

Switch on thermal cycler and wait for the instrument to initialize.

Place tubes in thermal cycler and run the conditions shown in Table 5.

A	B	C	D
Thermocycling conditions	Temperature (degrees celcius)	Time	Cycle(s)
cDNA synthesis	50	10 minutes	1
Pre-denaturation	98	2 minutes	1
Denaturation	98	10 seconds	40
Annealing	56	20 seconds
Extension	72	2 minutes
Final extension	72	10 minutes
Hold	4	∞	Hold

Table 5. One-step RT-PCR amplification conditions.

After PCR is complete, remove PCR tubes from thermal cycler and proceed to second-round PCR amplification, or store at -20°C.

Second-round PCR amplification.

In PCR amplification laboratory, remove second-round PCR master mix from-20°C.

Gently mix one-step RT-PCR amplicons by pipetting up and down, and add 2µL to the respective second-round PCR master mix tubes.

Gently tap PCR tubes and centrifuge briefly.

Place the PCR tubes in thermal cycler and run the conditions shown in Table 6.

A	B	C	D
Thermocycling conditions	Temperature (degrees celcius)	Time	Cycle(s)
Pre-denaturation	94	2 minutes	1
Denaturation	95	10 seconds	40
Annealing	56	20 seconds
Extension	72	2 minutes
Final extension	72	10 minutes	1
Hold	4	∞	Hold

Table 6. Second-round PCR amplification conditions.

After PCR is complete, remove PCR tubes from thermal cycler and proceed to run gel electrophoresis, or store second-round amplicons at -20°C.

Gel electrophoresis on agarose gel.

Assemble gel casting apparatus by securing gel casting gates on both sides of the casting tray. Place gel comb in upper slot of the casting tray.

Add 1µL of gel loading dye to a piece of parafilm paper for each amplicon to be loaded on the gel.

Mix with 5µL of each amplicon and load into respective wells of the agarose gel.

Mix 3µL of gel loading dye with 3µL of DNA ladder, and load into the first well of the agarose gel.

Close the lid of the electrophoresis tank ensuring that the negative electrode (black) is positioned close to the agarose gel wells.

Switch PowerPac on and set voltage at 120V.

Run the gel for at least 0h 45m 0s.

After running gel, remove it from electrophoresis tank and visualize on BioMetra UV detection unit.

PCR amplicons should be at a size of approximately 2.9 kilobases (kb) as measured against the DNA ladder in well position 1.

Proceed to PCR product purification or store amplicons at -20°C until use.

Prepare 1X TBE by adding 1 part 10X TBE buffer to 9 parts distilled water. For example to make 1L of 1X TBE, mix 100mLof 10X TBE with 900mL of distilled water.

Prepare 1% agarose gel by adding two 0.5g agarose tablets to 100mL of 1X TBE in a Schott bottle.

Heat in microwave for approximately 0h 2m 0s, swirling occasionally to ensure agarose tablets have dissolved completely.

Gently remove from microwave taking care not to spill over and allow cooling at room temperature, or by running the base of the flask under cold water.

Add 10µL of SYBR safe gel stain (10,000X concentrate) to 100mL gel.

Allow agarose gel to set (~0h 30m 0s).

Once set, carefully remove gel comb and the casting gates.

Place agarose gel in the electrophoresis tank with wells closest to the anode (i.e. negative electrode (black) position on top) and immerse in 1X TBE buffer.

PCR product purification using ExoSAP-IT PCR production purification

For samples with successful amplification, label PCR tubes required for purification.

Tap amplicon tubes gently to mix and briefly spin to bring the contents to bottom of tube .

Remove ExoSAP-IT Express reagent from -20°C freezer.

Gently tap ExoSAP-IT Express reagent to mix and briefly spin to bring contents to bottom of tube.

Mix 10µL of PCR amplicon with4µL of ExoSAP-IT Express reagent for a combined 14µL reaction volume.

Mix thoroughly by tapping the tube and quick spin to bring contents to the bottom of the tube .

Place the tubes in thermal cycler and run the conditions shown in Table 7.

A	B	C	D
Thermocycling conditions	Temperature (degrees celcius)	Time	Cycle(s)
Cleanup	37	4 minutes	1
Enzyme inactivation	80	1 minute	1
Hold	4	∞	Hold

Table 7. PCR purification conditions.

After ~0h 5m 0s on thermal cycler, purified PCR products are ready for DNA sequencing.

BigDye cycle sequencing.

The following procedure is used to set up the sequencing reaction mix using BigDye Terminator v3.1 cycle sequencing kit (LifeTechnologies) with 8 primers per sample.

Cover plate with aluminium plate seal, tap plate gently to mix, and spin plate briefly.

Remove aluminium plate seal and cover plate with PCR septa mat.

Place on thermal cycler and run the conditions shown in Table 10.

A	B	C	D
Thermocycling conditions	Temperature (degrees celcius)	Time	Cycle(s)
Pre-denaturation	96	1 minutes	1
Denaturation	96	10 seconds	35
Annealing	50	5 seconds
Extension	60	4 minutes
Hold	4	∞

Table 10. Cycle sequencing reaction conditions.

After the cycle sequencing reaction run is complete, proceed with BigDye sequencing reaction purification, or store the plate covered in foil at 4°Cfor up to 1 week, or at -20°Cfor storage longer than 1 week .

Remove SAM and XTerminator reagents from fridge (stored at 4°C) and equilibrate to room temperature prior to preparing SAM/BigDye XTerminator mix.

Following BigDye cycle sequencing reaction, centrifuge 96-well reaction plate at 4000rpm,0h 0m 0s for 0h 1m 0s.

Thoroughly vortex XTerminator solution for at least0h 0m 30s.

Prepare the SAM/BigDye XTerminator bead working solution in a 50mL Falcon tube, by adding reagent volumes shown in Table 11.

A	B
Component	Volume per 10μL reaction
SAM solution	45μL
BigDye XTerminator solution	10μL
Total volume	55μL

Mix the SAM/BigDye XTerminator bead working solution thoroughly by vortexing and dispense in a reagent reservoir.

Remove PCR septa mat from 96-well reaction plate and add 55µLof SAM/BigDye XTerminator bead working solution to each well.

Thaw all reagents at room temperature.

Cover plate with aluminium plate seal.

Vortex plate on a plate shaker at 1800rpm,0h 0m 0s for 0h 30m 0s.

After vortexing, centrifuge plate at 4000rpm,0h 0m 0s for 0h 2m 0s.

Label eight 1.5mL tubes, one for each primer mix.

Vortex sequencing buffer and primers and briefly spin to bring contents to the bottom of the tube.

Mix BigDye reagent by tapping tube and briefly spin to bring contents to bottom of tube.

Prepare master mix for each primer as shown in Table 8.

A	B	C
Reagent	Volume (µl)/ reaction	Concentration per reaction
Nuclease-free Water	6.1	-
5X Sequencing buffer	2	1X
Primer (3.2µM)	0.5	0.2µM
BigDye Sequencing mix	0.4	-
Total	9.0

Table 8. BigDye cycle sequencing reaction mix.

A	B	C	D
Primer (Direction)	Primer sequence	HXB2	Gene
RTC1F (Forward)	ACCTACACCTGTCAACATAATTG	2486 - 2508	PR and RT
RTC2R (Reverse)	TGTCAATGGCCATTGTTTAACCTTTGG	2630 - 2604	PR and RT
RTC3F (Forward)	CACCAGGGATTAGATATCAATATAATGTGC	2965 - 2994	PR and RT
RTC4R (Reverse)	CTAAATCAGATCCTACATACAAGTCATCC	3101 - 3129	PR and RT
KVL076 (Forward)	GCACAYAAAGGRATTGGAGGAAATGAAC	4161 - 4188	IN
KVL082 (Forward)	GGVATTCCCTACAATCCCCAAAG	4647 - 4669	IN
KVL083 (Reverse)	GAATACTGCCATTTGTACTGCTG	4750 - 4772	IN
PAN2R (Reverse)	CTGCCATCTGTTTTCCATAYTC	5037 - 5058	IN

Table 9. Details of sequencing primers.

Label 96-well plate for the cycle sequencing reaction PCR.

Dispense 9µL of each primer mix into the respective wells of a 96-well plate.

Add 1µL purified amplicons to the respective wells.

Capillary electrophoresis on 3730 Genetic Analyzer

Setup plate run on Genetic Analyzer (Applied Biosystems), according to cycle sequencing reaction plate layout.

Load plate onto Genetic Analyzer (Applied Biosystems) and start capillary electrophoresis run.

At the end of the run, remove the plate from the Genetic Analyzer and dispose in biohazard waste.

Login to SeqA6 software on a computer connected to the Genetic Analyzer and run primary analysis.

Save the analyzed data files, and copy the ab1 sequence files to USB disk.

Sequence editing and drug resistance interpretation.

Upload ab1 sequence files from USB key into Geneious software (Biomatters Ltd, New Zealand).

Trim and edit sequences for good quality reads.

Align all sequence files for each sample to an annotated HIV reference sequence, and edit sequences where necessary. This entails identifying any ambiguities, and verifying positions with mixed bases by inspecting quality (symmetry, height, and background) of the base calls.

Align all consensus sequences and perform phylogenetic tree reconstruction to assess sequence similarity.

Export consensus sequences in FASTA format and assess HIV drug resistance mutations using the Stanford University HIV drug resistance database (http://hivdb.stanford.edu/hivdb/by-sequences/).

• The following procedure is used to set up the sequencing reaction mix using the BigDye Terminator v3.1 cycle sequencing kit (LifeTechnologies) and 8 primers per sample.

• Thaw all reagents at room temperature.

• Label 1.5mL tubes, one for each primer mix.

• Vortex sequencing buffer and primers and briefly spin to bring the contents to the bottom of the tube.

• Mix BigDye by tapping tube and briefly spin to bring the contents to the bottom of the tube.

• Prepare master mix for each primer as shown in Table 4.

A	B	C
Reagent	Volume per reaction (µl)	Concentration per reaction
PCR grade water	6.1	-
5X Sequencing buffer	2	1.1X
Primer (3.2µM)	0.5	0.2µM
BigDye Sequencing mix	0.4	-
Total	9.0	-

Table 6 Sequencing reaction mix for each sequencing primer

A	B	C	D
Primer (Direction)	Primer sequence	HXB2	Gene
RTC1F (Forward)	ACCTACACCTGTCAACATAATTG	2486 - 2508	PR and RT
RTC2R (Reverse)	TGTCAATGGCCATTGTTTAACCTTTGG	2630 - 2604	PR and RT
RTC3F (Forward)	CACCAGGGATTAGATATCAATATAATGTGC	2965 - 2994	PR and RT
RTC4R (Reverse)	CTAAATCAGATCCTACATACAAGTCATCC	3101 - 3129	PR and RT
KVL076 (Forward)	GCACAYAAAGGRATTGGAGGAAATGAAC	4161 - 4188	IN
KVL082 (Forward)	GGVATTCCCTACAATCCCCAAAG	4647 - 4669	IN
KVL083 (Reverse)	GAATACTGCCATTTGTACTGCTG	4750 - 4772	IN
PAN2R (Reverse)	CTGCCATCTGTTTTCCATAYTC	5037 - 5058	IN
Optional primers
2586F (Forward)	AAGCCAGGAATGGATGGCCCA	2586 - 2606	PR and RT
2713R (Reverse)	GGATTTTCAGGCCCAATTTTTG	2713 - 2692	PR and RT
PAN3F (Forward)	TTAAAAGAAAAGGGGGGATTGGG	4783 - 4805	IN
KVL084 (Reverse)	TCCTGTATGCARACCCCAATATG	5243 - 5265	IN

Table 7 Sequencing primers for complete HIV-1 PR, RT and IN sequencing.

• Label 96 well plate for the sequencing reaction PCR.

• Dispense9µL of each mix into the respective wells in a 96 well plate.

• Add 1µL purified amplicons to the respective wells.

Note: Note: It is recommended to use the plate layout on the worksheet for cycle sequencing reaction and sequencing reaction purification.

• Details of the sequencing primers are shown in table 7.

  BigDye XTerminator Purification

Note: Note: Remove SAM and XTerminator reagents from fridge and equilibrate to room temperature prior to preparing SAM/BigDye XTerminator solution

• Centrifuge the reaction plate for 1 minute following the sequencing reaction.

• Ensure that the XTerminator solution is homogenized by vortexing thoroughly.

• Vortex the bottle of BigDye XTerminator beads for 0h 0m 8s to 0h 0m 10s before mixing with the SAM solution.

• Prepare the SAM/BigDye XTerminator bead working solution (as shown in Table 11) in a 50mLFalcon tube.

A	B	C
Component	Volume per 10μl reaction	Volume (µl) x n
SAM solution	45μL
BigDye XTerminator bead solution	10μL
Total volume	55μL

Table 8 SAM/BigDye XTerminator solution

• Mix the SAM/BigDye XTerminator bead working solution thoroughly by vortexing and dispense in a reagent reservoir.

• Remove the PCR septa mat and add the required volume (55µL/ 110µL) of SAM/BigDye XTerminator bead working solution using a multi-channel pipette.

• Seal the plate using an aluminium plate seal.

• Vortex the plate on a plate shaker at 1800rpm for 0h 30m 0s

• Centrifuge the plate at 4000rpmfor 0h 4m 0s

• Sequence samples on an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, United States)