TITLE: FL-BEEHIV HIV-1 Genotyping and Drug Resistance by Next Generation Sequencing-High throughput extraction
AUTHORS: Brenna M McGruder Rawson, Jason Blanton

[DESCRIPTION]
The Florida Department of Health's Bureau of Public Health Laboratories in Jacksonville is developing a protocol for the Next Generation Sequencing (NGS) of HIV, primarily for the purpose of drug-resistant mutation identification. This HIV-1 protocol uses amplicon based sequencing based on primers designed by the BEEHIVE Consortium (https://www.beehive.ox.ac.uk/). The amplified pol gene regions are used in both genotyping and drug resistance determination.   Our protocol utilizes newer enzymes with higher fidelity for sequencing and Illumina sequencing technology.  The NGS data generated can also be used in surveillance and outbreak monitoring, giving epidemiologist more information about circulating viral genomes.  There is also the potential that this protocol can be expanded to whole genome sequencing for HIV-1.
The imminent sunsetting of ViroSeq (Abbott Molecular) has required many labs to look for new methods to continue identifying HIV-1 drug resistance strains for both clinical management and epidemiological study. NGS was chosen as it is more cost effective than investing in a single pathogen platform. NGS allows for one sample to produce results and data that can aid not just a patient but an entire population. 

[STEPS]
SECTION: RNA Extraction
1. Extract RNA using the Thermofisher MagMAX Viral and Pathogen(MVP) Nucleic Acid Isolation Kit I or MVPII using the Kingfisher Flex
- The difference is that the MVPI is Reasearch Use Only (RUO) and the MVPII is In Vitro Diagnostic (IVD) approved
-Both kits are the same, however the MVPI kit is able to be purchased in a smaller format, whereas the MVPII kit is only available in 1,000 or 2,000 reactions. 
SECTION: RNA Extraction
1.1. Prepare the following 4 plates for the appropriate number of sample/controls (Following the Procedures for viral nucleic acid isolation- 200uL sample volume):
1. Wash Solution 1 plate- 500uL of MVP Wash Solution to each well
2. Wash Solution 2 plate- 1000uL of freshly prepared 80% Ethanol
3. Elution plate- 50uL of MVP Elution Solution 
4. Sample Plate
     a. Add 5.0uL Proteinase K to each sample/control well
     b. Add 200uL of sample to designated well
     c. Add 275uL of Binding Bead solution to each sample/control well 
          -Binding Bead mix is 265uL Binding Solution + 10uL Magnetic Beads per sample (ie 100uL beads+         
            2.65mL Binding solution)
         - Do NOT vortex the Binding Bead solution- only mix well by inversion
      


SECTION: RNA Extraction
1.2. Run the program on the KingFisher Flex (200uL)
SECTION: cDNA Synthesis
2. Master Mix
4.0 µL 
6.0 µL 
10.0 µL 

SECTION: cDNA Synthesis
3. Run the following protocol on a thermocyler
25 °C 10 min 
50 °C 10 min 
85 °C 5 min 

SECTION: Amplicon PCR
4. Each fragment will need to be amplified in an individual PCR reaction
Set 1
Pan-HIV-1_2F     GGG AAG TGA YAT AGC WGG AAC 
Pan-HIV-1_2R      CTG CCA TCT GTT TTC CAT ART C 

Set 2
Pan-HIV-1_3F      TTA AAA GAA AAG GGG GGA TTG GG 
Pan-HIV-1_3R      TGG CYT GTA CCG TCA GCG 
SECTION: Amplicon PCR
5. Run the following protocol on a thermocycler
105 °C 
50 °C 30 s 
98 °C 30 s 
    35 cycles 
72 °C 5 min 
4 °C 



SECTION: Amplicon PCR
4.1. Master Mix
12.5 µL 
0.5 µL 20 micromolar (µM) 
0.5 µL 20 micromolar (µM) 
6.5 µL 
5.0 µL 
SECTION: Amplicon PCR
6. Bead clean up using a ratio of 0.5-  follow the AMPure XP bead protocol for PCR purification


SECTION: Amplicon PCR
7. Check fragment on Tapestation or gel
SECTION: Fragment Normalizing and Pooling
8. Fragments can be pooled in eqimolar amounts or in equal concentrations.  



SECTION: Fragment Normalizing and Pooling
8.1. Pool fragments
SECTION: Fragment Normalizing and Pooling
8.2. Dilute as needed to achieve 1.0 ng  input concentration for library preparation

SECTION: Library Prep
9. Follow Illumina Protocol for Nextera XT DNA Library Sample Prep
SECTION: Pooling Samples
10. Amplicon quality can effect how many samples can be pooled onto one run.  Use caution in deciding how many samples to pool.  


SECTION: Amplicon PCR
7.1. Band size should be
Amplicon 1- 3.5 kB
Amplicon 2- 3.0 kB
SECTION: Analysis
11. We currently use SmartGene HIV-1 pipeline (https://www.smartgene.com/) for analysis
Pipeline Name: HIV-1 PR+RT+IN 
Version 2.2.0HIV1 V1.6
Noise Filter [%] 0.5
Interpretation cut off [%] 5.0


Minimum read depth and additional criteria should be determined by your institution
SECTION: References
12. Gall A, Ferns B, Morris C, Watson S, Cotten M, Robinson M, Berry N, Pillay D, Kellan P. Universal Amplification, Next-Generation Sequencing, and Assembly of HIV-1 Genomes. Journal of Clinical Microbiology. 2012; 50:12. 
doi: 10.1128/JCM.01516-12

Cornelissen M, Gall A, Vink M, Zorkrager F, Binter S, Edwards S, Jurriaans S, Bakker M, Ong SH, Gras L, van Sighem A, Bexemer D, de Wolf F, Reiss P, Kellam P, Berkhout B, Fraser C, van der Kuyl AC, the BEEHIVE Consortium. From clinical samples to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing. Virus Research. 2017; 239:10-16. doi: 10.1016/j.virusres.2016.08.004