TITLE: HIV-1 Genotyping and Drug Resistance by Next Generation Sequencing
AUTHORS: Brenna McGruder Rawson, Matthew  Schimenti, 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. The amplified </span><span style = "font-style:italic;">pol</span><span> gene regions are used in both genotyping and drug resistance determination. The NGS data generated can also be used in surveillance and outbreak monitoring, giving epidemiologist more information about circulating viral genomes.</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)
?. [Reverse-Transcription and Amplification]
Master Mix with Invitrogen SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA Polymerase (cat 12574-035)
[2x Reaction Mix]
[HIV2252-F]
[HIV5073-R]
[SuperScript III Platinum Taq]
[DMSO]
[viral RNA template]
?. [Second Amplification]
Master Mix with Invitrogen SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA Polymerase (cat 12574-035)
[2x Reaction Mix]
[FL_HIV_Ext Nest FW]
[FL_HIV_Ext Nest RV]
[SuperScript III Platinum Taq]
[DMSO]
[clean cDNA template]
?. [Library Preperation]
Quantitate Samples and dilute as needed to achieve  input concentration per sample
1 ng
?. [Pooling Samples]
Amplicon quality can effect how many samples can be pooled onto one run.  Use caution in deciding how many samples to pool.
?. [Analysis]
We currently use SmartGene HIV-1 pipeline (https://www.smartgene.com/) for analysisPipeline Name: HIV-1 PR+RT+INVersion 2.2.0HIV1 V1.6Noise Filter [%] 0.5Interpretation cut off [%] 5.0Minimum read depth and additional criteria should be determined by your institution
?. [References]
Dudley DM, Bailey AL, Mehta SH, Hughes AL, Kirk GD, Westergaard RP, O'Connor DH. Cross-clade simultaneous HIV drug resistance genotyping for reverse transcriptase, protease, and integrase inhibitor mutations by Illumina MiSeq. Retrovirology. 2014 Dec 23;11:122. doi: 10.1186/s12977-014-0122-8. PMID: 25533166; PMCID: PMC4302432.
?. [RNA Extraction]
Add
[carrier RNA/ sample]
?. [RNA Extraction]
Follow Kit Instructions
?. [Reverse-Transcription and Amplification]
Reverse-Transcription/Amplification PCR thermocycler settings15 cyclesFinal extension
[Lid setting]
50 °C
94 °C
55 °C
68 °C
78 °C
[Hold]
?. [Reverse-Transcription and Amplification]
Using a ratio of 0.5  follow the AMPure XP bead protocol for PCR purification
?. [Second Amplification]
Second Amplification PCR thermocycler settings40 cyclesFinal extension
[Lid setting]
94 °C
55 °C
68 °C
78 °C
[Hold]
?. [Second Amplification]
Using a ratio of 0.5  follow the AMPure XP bead protocol for PCR purification
?. [Second Amplification]
Visualize samples using Tapestation or agarose gel
?. [Second Amplification]
Fragments should be ~2.9 kB
?. [Second Amplification]
If the 2.9 kb fragment is isolated then samples should sequence well.  If there are additional fragments sequencing is still possible, but samples may require a higher input concentration to achieve desired coverage
?. [Library Preperation]
Follow Illumina Protocol for Nextera XT DNA Library Sample Prep
?. [Pooling Samples]
Normalize samples prior to pooling by dilution
?. [Pooling Samples]
Samples should be pooled in an equal volume amount.  If desired the pool can be cleaned using an additional AMPure XP bead clean.
?. [Pooling Samples]
Dilute the Pooled Library to apx. 0.07 ng/µL or a concentration found in the Illumina iSeq manual
?. [Pooling Samples]
Quantitate the Pooled Library
?. [Pooling Samples]
Add  to the pool
5 µl
[PhiX]
?. [Pooling Samples]
Load according to Illumina instructions