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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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 (</span><a href="https://www.beehive.ox.ac.uk" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.beehive.ox.ac.uk/</span></a><span>). 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.</span></div><div class = "text-block">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. </div></div>

[STEPS]
?. [RNA Extraction]
Extract RNA using the Qiagen Viral RNA Mini Kit (DSP or RUO)
?. [RNA Extraction]
Add   per sample
[carrier RNA]
?. [RNA Extraction]
Follow Kit instructions for extraction
?. [cDNA Synthesis]
Master Mix
[SuperScript IV VILO Master Mix]
[Nuclease Free Water]
[RNA template]
?. [cDNA Synthesis]
Run the following protocol on a thermocyler
25 °C
50 °C
85 °C
?. [Amplicon PCR]
Each fragment will need to be amplified in an individual PCR reactionSet 1Pan-HIV-1_2F     GGG AAG TGA YAT AGC WGG AAC Pan-HIV-1_2R      CTG CCA TCT GTT TTC CAT ART C Set 2Pan-HIV-1_3F      TTA AAA GAA AAG GGG GGA TTG GG Pan-HIV-1_3R      TGG CYT GTA CCG TCA GCG
?. [Amplicon PCR]
Run the following protocol on a thermocycler    35 cycles
[Lid]
50 °C
98 °C
72 °C
[Hold]
?. [Amplicon PCR]
Master Mix
[2x Q5 Master Mix]
[Forward Primer]
[Reverse Primer]
[Nuclease Free Water]
[cDNA template]
?. [Amplicon PCR]
Bead clean up using a ratio of 0.5-  follow the AMPure XP bead protocol for PCR purification
?. [Amplicon PCR]
Check fragment on Tapestation or gel
?. [Fragment Normalizing and Pooling]
Fragments can be pooled in eqimolar amounts or in equal concentrations.
?. [Fragment Normalizing and Pooling]
Pool fragments
?. [Fragment Normalizing and Pooling]
Dilute as needed to achieve   input concentration for library preparation
1 ng
?. [Library Prep]
Follow Illumina Protocol for Nextera XT DNA Library Sample Prep
?. [Pooling Samples]
Amplicon quality can effect how many samples can be pooled onto one run.  Use caution in deciding how many samples to pool.
?. [Amplicon PCR]
Band size should beAmplicon 1- 3.5 kBAmplicon 2- 3.0 kB
?. [Analysis]
We currently use SmartGene HIV-1 pipeline (https://www.smartgene.com/) for analysisPipeline Name: HIV-1 PR+RT+IN Version 2.2.0HIV1 V1.6Noise Filter [%] 0.5Interpretation cut off [%] 5.0Minimum read depth and additional criteria should be determined by your institution
?. [References]
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.01516-12Cornelissen 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