TITLE: Next Generation Sequencing of HIV-1 Drug Resistant Mutations
AUTHORS: Brenna M McGruder Rawson

[DESCRIPTION]
The Florida Department of Health's Bureau of Public Health Laboratories in Jacksonville has developed 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 can be used in both genotyping and drug resistance determination.   Our protocol utilizes newer enzymes with higher fidelity for sequencing and Illumina sequencing technology.  We have cross verified 3 different Illumina Sequencing platforms to ensure that all produce equivalent results so that in the event of a surge samples can be sequenced quickly and in mixed-species pools. 
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. 

[BEFORE_START]
We are happy to share HIV-1 samples for public health lab validations if we have materials available. 

[GUIDELINES]
All Lab Developed Tests are still subject to CLIA.  Please consult with your CLIA director to establish an appropriate study to develop your own HIV-1 sequencing test.

[STEPS]
SECTION: RNA Extraction
1. RNA Extraction has been verified using the following methods
SECTION: RNA Extraction
1.1.  Qiagen QIAmp Viral RNA Mini Kit (DSP or RUO)
https://www.qiagen.com/us/products/diagnostics-and-clinical-research/sample-processing/qiaamp-viral-rna-kits/
SECTION: RNA Extraction
1.2. Thermofisher MagMAX Viral/Pathogen II (MVP II) Nucleic Acid Isolation kit
https://www.thermofisher.com/order/catalog/product/A48383
SECTION: cDNA Synthesis
2. Master Mix
4.0 µLSuperScript IV VILO Master Mix
6.0 µL Nuclease Free Water
10.0 µL RNA template

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: cDNA Synthesis
3. Run the following protocol on a thermocyler
25 °C10 min
50 °C10 min
85 °C5 min

SECTION: Amplicon PCR
5. PCR
The two primers do have different optimal annealing temperatures, but we have found that they both can be run at the same temperature.

105 °C Lid
4 °C 
55 °C30 s
98 °C30 s
    35 cycles
72 °C5 min
4 °CHold

SECTION: Amplicon PCR
4.1. Master Mix

12.5 µL 2x Q5 Master Mix
0.5 µL Forward Primer20 Micromolar (µM)
0.5 µL Reverse Primer20 Micromolar (µM)
6.5 µL Nuclease Free Water
5.0 µL cDNA template

6. Bead clean up using a ratio of 0.5-  follow the AMPure XP bead protocol for PCR purification.
7. Check fragment on Tapestation or gel.  

Band size should be
Amplicon 1- 3.5 kB
Amplicon 2- 3.0 kB
SECTION: Sample Amplicon Pooling
8. Sample fragments 1 and 2 can be pooled in eqimolar amounts or in equal concentrations.

For HIVdb version9.0(last updated on2021-02-22) Primer Set 1 usually has sufficient coverage.  Primer Set 2 offers end coverage.
SECTION: Sample Amplicon Pooling
9. Pool fragments
SECTION: Sample Amplicon Pooling
10. Dilute as needed to achieve 1.0 ng input concentration for library preparation

SECTION: Library Prep
11. Follow Illumina Protocol for Nextera XT DNA Library Sample Prep
SECTION: Library Pooling
12. Amplicon quality can effect how many samples can be pooled onto one run.  Use caution in deciding how many samples to pool.  
SECTION: Analysis
14. We currently use SmartGene HIV-1 pipeline (https://www.smartgene.com/) for analysis

Pipeline Name: HIV-1 PR+RT+IN 
Version 2.4.5_HIV1_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
15. 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

Previous Protocols
https://dx.doi.org/10.17504/protocols.io.btrnnm5e
https://dx.doi.org/10.17504/protocols.io.btpqnmmw
SECTION: Sequencing
13. We have successfully sequenced these libraries on the following platforms:

iSeq
MiSeq
NextSeq


The MiSeq and NextSeq are usually mixed organism pools.  This has had no discernable adverse effect on HIV-1 Drug-Resistance Sequencing results.