TITLE: Affordable method for genotyping HIV-1 reverse transcriptase, protease and integrase genes: an in-house protocol
AUTHORS: Sontaga Manyana, Melendhran Pillay,  Lilishia Gounder,  Aabida Khan,  Pravi Moodley, Kogieleum Naidoo,  Benjamin Chimukangara

[DESCRIPTION]
HIV drug resistance (HIVDR) remains a major threat to achieving sustainable viral suppression on antiretroviral treatment. To overcome concerns over increasing levels in pretreatment drug resistance and subsequent cost implications, most countries including those in resource limited settings (RLS) adopted use of dolutegravir (DTG), a potent integrase strand transfer inhibitor (INSTI). As more people receive INSTIs, the demand for INSTI resistance testing is increasing. Current HIVDR testing methods in RLS focus on genotyping HIV protease (PR) and reverse transcriptase (RT) genes, separate from the integrase (IN) gene. However, such an approach is expensive and evidently increases the workload for HIVDR genotyping. As a result, affordable and labour efficient methods that genotype all relevant HIV-1 genes (i.e., the PR, RT and IN genes) are required to guide clinical decisions, especially in RLS where cost is a major limiting factor. Therefore, we developed a protocol for genotyping complete HIV-1C genes at an affordable cost and time-efficient manner, that can be adopted for use in HIV drug resistance genotyping. The main limitation to this protocol is that it was only tested on HIV-1C samples, and thus might not be effective against other HIV subtypes. In conclusion, the expected results of using this protocol include complete HIV-1C sequence coverage of all relevant viral gene regions; i.e. PR codons 1 - 99, RT codons 1 - 560, and IN codons 1 - 288, for HIVDR genotyping.



[BEFORE_START]
Ensure that the working areas are disinfected 

Remember to check the expiring date of the reagents before use

Ensure that instruments maintenance is up to date

[GUIDELINES]
Introduction
This protocol describes a simple, labour efficient and affordable genotyping method for detecting HIVDR mutations in the HIV-1C protease, reverse transcriptase and integrase genes. This method was produced at the Department of Virology National Laboratory Services, University of KwaZulu-Natal, in Durban, South Africa. 

Briefly, a simple and innovative single assay polymerase chain reaction (PCR) technique was designed to amplify an approximate 2.9kb HIV-1 pol gene. The method was designed to genotype HIV-1 pol genes with complete coverage of all regions with known HIV drug resistance mutations by overlapping 8 sequencing primers. The amplicons generated are sequenced on an ABI Genetic Analyzer, with HIVDR mutations determined using the Stanford HIV drug resistance database.

Purpose
The purpose of this protocol is to provide detailed instructions that should be followed when genotyping HIV-1 pol genes from plasma samples using BigDye Terminator v3.1 cycle sequencing kit (LifeTechnologies) and Sanger sequencing. 

Competing Interests
There are no competing interests to declare.

Data availability
https://www.ncbi.nlm.nih.gov/nuccore/OM468298


[STEPS]
SECTION: PCR MASTER MIX
2. Preparation of one-step reverse transcription and second-round PCR master mix 
SECTION: DETECTION OF AMPLICONS
6. Gel electrophoresis on agarose gel.
SECTION: SEQUENCE ANALYSIS
10. Sequence editing and drug resistance interpretation.
SECTION: EXTRACTION
1. Sample preparation and RNA extraction on NucliSENS easyMAG 
SECTION: EXTRACTION
1.1. Retrieve plasma samples from -80 °C freezer and leave to equilibrate to room temperature prior to processing.
SECTION: EXTRACTION
1.12. Load NucliSENS easyMAG consumables on to the NucliSENS easyMAG instrument and scan barcodes on consumables.
SECTION: PCR MASTER MIX
2.2. All reagents except the enzymes (Superscript IV and Platinum Taq DNA Polymerase) must be thawed and vortexed for 5 s  to  10 s
 
SECTION: PCR MASTER MIX
2.3. Label reverse transcription and second-round PCR tubes  0.2 mL with sample numbers, accordingly.
SECTION: PCR MASTER MIX
2.4. Label two 1.5 mL microcentrifuge tubes as follows; Tube 1: for RT one-step PCR master mix, and Tube 2: for second-round PCR master mix. 



 

 

SECTION: PCR MASTER MIX
2.5. 
Prepare RT-PCR master mix as shown in Table 1, using Superscript IV one-step PCR reagents.

     Reagent         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        -          Thermocycling conditions                   Temperature (oC)        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        1           Hold        4        ∞        Hold     


 

      Name       Sequence      Direction      HXB2 position         PANA2AF       GAGGCAATGAGCCAARCAAACA         Forward      1882   ---- 1903            PANA3AR       TTCCAGGGCTCTAGKTTAGG      Reverse      5846   ---- 5865   

 
 

 
SECTION: DETECTION OF AMPLICONS
6.1. 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.
SECTION: DETECTION OF AMPLICONS
6.2. 
Prepare 1X TBE by adding 1 part 10X TBE buffer to 9 parts distilled water. For example to make 1 L of 1X TBE, mix 100 mLof 10X TBE with 900 mL of distilled water.

SECTION: DETECTION OF AMPLICONS
6.3. 
Prepare 1% agarose gel by adding two 0.5 g agarose tablets to 100 mL of 1X TBE in a Schott bottle.

 

SECTION: DETECTION OF AMPLICONS
6.4. Heat in microwave for approximately 2 min, swirling occasionally to ensure agarose tablets have dissolved completely. 

 

SECTION: DETECTION OF AMPLICONS
6.5. 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.

 

SECTION: DETECTION OF AMPLICONS
6.6. Add 10 µL of SYBR safe gel stain (10,000X concentrate) to 100 mL gel. 
 


SECTION: DETECTION OF AMPLICONS
6.7. Allow agarose gel to set (~30 min).
SECTION: DETECTION OF AMPLICONS
6.8. Once set, carefully remove gel comb and the casting gates. 

SECTION: DETECTION OF AMPLICONS
6.9. 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.

SECTION: DETECTION OF AMPLICONS
6.10. Add 1 µL of gel loading dye to a piece of parafilm paper for each amplicon to be loaded on the gel.


SECTION: DETECTION OF AMPLICONS
6.11. Mix with 5 µL of each amplicon and load into respective wells of the agarose gel.
 


SECTION: DETECTION OF AMPLICONS
6.12. Mix 3 µL of gel loading dye with 3 µL of DNA ladder, and load into the first well of the agarose gel.

SECTION: DETECTION OF AMPLICONS
6.13. Close the lid of the electrophoresis tank ensuring that the negative electrode (black) is positioned close to the agarose gel wells. 
SECTION: DETECTION OF AMPLICONS
6.14. Switch PowerPac on and set voltage at 120V.
SECTION: DETECTION OF AMPLICONS
6.15. Run the gel for at least 45 min.


 


SECTION: DETECTION OF AMPLICONS
6.16. After running gel, remove it from electrophoresis tank and visualize on BioMetra UV detection unit.
SECTION: DETECTION OF AMPLICONS
6.17. PCR amplicons should be at a size of approximately 2.9 kilobases (kb) as measured against the DNA ladder in well position 1.

 

SECTION: PCR PRODUCT PURIFICATION
7.6. Mix thoroughly by tapping the tube and quick spin to bring contents to the bottom of the tube. 
SECTION: PCR PRODUCT PURIFICATION
7.5. Mix 10 µL of PCR amplicon with4 µL of ExoSAP-IT Express reagent for a combined 14 µL reaction volume. 

SECTION: PCR PRODUCT PURIFICATION
7.4. Gently tap ExoSAP-IT Express reagent to mix and briefly spin to bring contents to bottom of tube.
SECTION: PCR PRODUCT PURIFICATION
7.3. Remove ExoSAP-IT Express reagent from -20 °C freezer.

SECTION: PCR PRODUCT PURIFICATION
7.2. Tap amplicon tubes gently to mix and briefly spin to bring the contents to bottom of tube.


 

SECTION: PCR PRODUCT PURIFICATION
7.1. For samples with successful amplification, label PCR tubes required for purification.
SECTION: PCR PRODUCT PURIFICATION
7. PCR product purification using ExoSAP-IT PCR production purification

SECTION: SEQUENCE ANALYSIS
10.1.  Upload ab1 sequence files from USB key into Geneious software (Biomatters Ltd, New Zealand). 
SECTION: SEQUENCE ANALYSIS
10.4. Align all consensus sequences and perform phylogenetic tree reconstruction to assess sequence similarity.
 

SECTION: SEQUENCE ANALYSIS
10.5. 
Export consensus sequences in FASTA format and assess HIV drug resistance mutations using the Stanford University HIV drug resistance database (https://hivdb.stanford.edu/hivdb/by-sequences/).
SECTION: BIGDYE CYCLE SEQUENCING v3.1
110. 
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 1.5 mL 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.


     Reagent      Volume (µl)/ reaction      Volume (µl) x n        PCR grade water      6.1               5X Sequencing buffer      2               Primer (3.2µM)      0.5               BigDye Sequencing mix      0.4               Total      9.0          

 
     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    
 
Label 96 well plate for the sequencing reaction PCR.

Dispense9 µL of each mix into the respective wells in a 96 well plate.

Add 1 µL purified amplicons to the respective wells. 

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: 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 8 s to 10 s before mixing with the SAM solution.

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

 
     Component       Volume per 10μL reaction      Volume per 20μL reaction        SAM solution       45μL      90μL        BigDye XTerminator bead solution       10μL      20μL        Total volume       55μL      110μL   


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 (55 µL/ 110 µL) 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 30 min

Centrifuge the plate at 4000rpmfor 4 min

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



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

SECTION: EXTRACTION
1.3. Mark the top of each tube on the outside and spin at 23000 x g for 60 minat 4 °C.

SECTION: EXTRACTION
1.5. Proceed to extraction room


 

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

 

SECTION: EXTRACTION
1.6. Pipette mix the 200 µL pellet and transfer to400 µL of lysis buffer.

SECTION: EXTRACTION
1.7. Vortex briefly and incubate at room temperature for 20 min. 

SECTION: EXTRACTION
1.8. While the sample is lysing, label disposable NucliSENS easyMAG consumables according to sample list.
SECTION: EXTRACTION
1.9. After incubation, transfer lysate (~600 µL) into respective wells of NucliSENS easyMAG consumables. 

SECTION: EXTRACTION
1.10. Vortex magnetic silica briefly and add 50 µL to numbered NucliSENS easyMAG consumables. 

SECTION: EXTRACTION
1.11. 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.
SECTION: EXTRACTION
1.13. Program NucliSENS easyMAG instrument and select 25 µL elution volume.

SECTION: EXTRACTION
1.14.  Start extraction run.

 

SECTION: EXTRACTION
1.15. After extraction is complete, transfer eluent into appropriately labelled1.5 mL microcentrifuge tubes.

SECTION: EXTRACTION
1.16. Discard NucliSENSeasyMAG consumables appropriately as biohazard waste.
SECTION: EXTRACTION
1.17. Proceed to perform PCR amplification or store RNA at -80 °C until use.

SECTION: SECOND-ROUND PCR
5. Second-round PCR amplification.
SECTION: SECOND-ROUND PCR
5.1. In PCR amplification laboratory, remove second-round PCR master mix from-20 °C.

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


 

SECTION: SECOND-ROUND PCR
5.3. Gently tap PCR tubes and centrifuge briefly.
SECTION: SECOND-ROUND PCR
5.4. Place the PCR tubes in thermal cycler and run the conditions shown in Table 6.
 
     Thermocycling Conditions               Temp      Time       Cycle(s)         Pre-denaturation      94      2min      1         Denaturation      95      10sec      40         Annealing      56      20sec         Extension      72      2min         Final extension      72      10min      1         Hold      4      ∞      Hold   
 
 

 
SECTION: PCR MASTER MIX
2.1. Proceed to PCR master mix room (i.e. clean room).

2.6. 
Prepare master mix for the second-round PCR as shown in Table 3, using Platinum Taq PCR reagents.

     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      -   
 
 Details of primers for second-round PCR master mix are shown in Table 4.
      Name       Sequence      Direction      HXB2 Position         Pro1       TAGAGCCAACAGCCCCACCA      Forward      2147   ---- 2166            5066R       ATCATCACCTGCCATCTGTTTTCCAT      Reverse      5041   ---- 5066   
 
2.7. Mix one-step RT-PCR master mix by pipetting up and down (or by gently tapping the tube), and transfer 20 µLto the respective 0.2 µL PCR tubes.
2.9. Take aliquoted one-step RT-PCR master mix into the extraction laboratory (or dead air space cabinet in general laboratory).

 

2.8. Mix second-round PCR master mix by pipetting up and down (or by gently tapping the tube), and transfer 23 µLto the respective 0.2 µL  PCR tubes.
SECTION: ADDING RNA
3. Adding RNA to one-step RT-PCR master mix.
SECTION: ADDING RNA
3.3. Add 5 µL RNA to respective tubes containing master mix.

 


SECTION: ADDING RNA
3.1. Proceed to extraction laboratory, or dead air space cabinet.
SECTION: ADDING RNA
3.4. Proceed to the PCR amplification room.
SECTION: ONE-STEP RT-PCR
4.4. After PCR is complete, remove PCR tubes from thermal cycler and proceed to second-round PCR amplification, or store at -20 °C.
SECTION: ONE-STEP RT-PCR
4.3. Place tubes in thermal cycler and run the conditions shown in Table 5.

     Thermocycling Conditions               Temp      Time       Cycle(s)         cDNA   synthesis      50      10min      1         Pre-denaturation      98      2min      1         Denaturation      98      10sec      40         Annealing      56      20sec         Extension      72      2min         Final extension      72      10min         Hold      4      ∞      Hold   

 

SECTION: ONE-STEP RT-PCR
4.2. Switch on thermal cycler and wait for the instrument to initialize.
SECTION: ONE-STEP RT-PCR
4.1. Gently tap PCR tubes to mix and centrifuge briefly.
SECTION: ONE-STEP RT-PCR
4. RT-PCR amplification process.
SECTION: SECOND-ROUND PCR
5.5. After PCR is complete, remove PCR tubes from thermal cycler and proceed to run gel electrophoresis, or store second-round amplicons at -20 °C.

SECTION: PCR PRODUCT PURIFICATION
7.7. Place the tubes in thermal cycler and run the conditions shown in Table 7.

     Thermocycling Conditions               Temp      Time       Cycle(s)        Cleanup      37      4 min      1        Enzyme inactivation      80      1 min      1        Hold      4      ∞      Hold   

SECTION: PCR PRODUCT PURIFICATION
7.8. After ~5 min on thermal cycler, purified PCR products are ready for DNA sequencing. 


 

 
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8. BigDye cycle sequencing.
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.1. 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.
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.2. Thaw all reagents at room temperature.
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.3. Label eight 1.5 mL tubes, one for each primer mix.

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.4. Vortex sequencing buffer and primers and briefly spin to bring contents to the bottom of the tube.
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.5. Mix BigDye reagent by tapping tube and briefly spin to bring contents to bottom of tube.


 

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.6. Prepare master mix for each primer as shown in Table 8.

     Reagent      Volume (µl)/ reaction      Volume (µl) x n        PCR grade water      6.1               5X Sequencing buffer      2               Primer (3.2µM)      0.5               BigDye Sequencing mix      0.4               Total      9.0          
  


 


     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    
 
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.7. Label 96-well plate for the cycle sequencing reaction PCR.
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.8. Dispense 9 µL of each primer mix into the respective wells of a 96-well plate.

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.12. Place on thermal cycler and run the conditions shown in Table 10.

     Thermocycling Conditions               Temp      Time       Cycle(s)         Pre-denaturation      96      1 min      1         Denaturation      96      10 sec      35         Annealing      50      5 sec         Extension      60      4 min         Hold      4      ∞      Hold   

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.9. Add 1 µL purified amplicons to the respective wells. 

 


SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.10. Cover plate with aluminium plate seal, tap plate gently to mix, and spin plate briefly.
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.16. Thoroughly vortex XTerminator solution for at least30 s.

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.11. Remove aluminium plate seal and cover plate with PCR septa mat.
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.13. 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.

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.14. Remove SAM and XTerminator reagents from fridge (stored at 4 °C) and equilibrate to room temperature prior to preparing SAM/BigDye XTerminator mix. 

 

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.15. Following BigDye cycle sequencing reaction, centrifuge 96-well reaction plate at 4000 rpm for 1 min.

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.17. Prepare the SAM/BigDye XTerminator bead working solution in a 50 mL Falcon tube, by adding reagent volumes shown in Table 11.
 
     Component       Volume per 10μL reaction      Volume per 20μL reaction        SAM solution       45μL      90μL        BigDye XTerminator bead solution       10μL      20μL        Total volume       55μL      110μL   
 
 

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.18. Mix the SAM/BigDye XTerminator bead working solution thoroughly by vortexing and dispense in a reagent reservoir.
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.19. Remove PCR septa mat from 96-well reaction plate and add 55 µLof SAM/BigDye XTerminator bead working solution to each well. 

 

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.20. Cover plate with aluminium plate seal.
SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.21. Vortex plate on a plate shaker at 1800 rpm for 30 min.

SECTION: CYCLE SEQUENCING REACTION AND PURIFICATION
8.22. After vortexing, centrifuge plate at 4000 rpm for 2 min.

 

SECTION: ADDING RNA
3.2. Thaw RNA, gently tap tubes, and briefly spin.
SECTION: DETECTION OF AMPLICONS
6.18. Proceed to PCR product purification or store amplicons at -20 °C until use.

SECTION: SEQUENCE ANALYSIS
10.2. Trim and edit sequences for good quality reads. 
 

SECTION: SEQUENCE ANALYSIS
10.3. 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. 
SECTION: CAPILLARY ELECTROPHORESIS
9. Capillary electrophoresis on 3730 Genetic Analyzer
SECTION: CAPILLARY ELECTROPHORESIS
9.1.  Setup plate run on Genetic Analyzer (Applied Biosystems), according to cycle sequencing reaction plate layout.
SECTION: CAPILLARY ELECTROPHORESIS
9.2. Load plate onto Genetic Analyzer (Applied Biosystems) and start capillary electrophoresis run.


SECTION: CAPILLARY ELECTROPHORESIS
9.3. At the end of the run, remove the plate from the Genetic Analyzer and dispose in biohazard waste.
SECTION: CAPILLARY ELECTROPHORESIS
9.5. Save the analyzed data files, and copy the ab1 sequence files to USB disk. 
SECTION: CAPILLARY ELECTROPHORESIS
9.4. Login to SeqA6 software on a computer connected to the Genetic Analyzer and run primary analysis.