Next Generation Sequencing of HIV-1 Drug Resistant Mutations
Brenna M McGruder Rawson
HIV-1
Drug resistance
Drug resistance mutations
Next generation sequencing
SmartGene
FLDOH
Retrovirus sequencing
HIV
public health
antiretroviral drugs
HIV-1 clinical test
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Abstract
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.
Steps
RNA Extraction
RNA Extraction has been verified using the following methods
Qiagen QIAmp Viral RNA Mini Kit (DSP or RUO)
Thermofisher MagMAX Viral/Pathogen II (MVP II) Nucleic Acid Isolation kit
cDNA Synthesis
Master Mix
4.0µLSuperScript IV VILO Master Mix
6.0µL Nuclease Free Water
10.0µL RNA template
Run the following protocol on a thermocyler
25°C``0h 10m 0s
50°C``0h 10m 0s
85°C``0h 5m 0s
Amplicon PCR
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
Master Mix
12.5µL 2x Q5 Master Mix
0.5µL Forward Primer20 Micromolar (µM)
0.5µL Reverse Primer20 Micromolar (µM)
6.5µL Nuclease Free Water
5.0µL cDNA template
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
50°C 0h 0m 30s
98°C 0h 0m 30s
40 cycles-
`98°C` `0h 0m 15s`
`55°C``0h 0m 30s`
`98°C``0h 0m 30s`
72°C``0h 5m 0s
4°CHold
Bead clean up using a ratio of 0.5- follow the AMPure XP bead protocol for PCR purification.
Check fragment on Tapestation or gel.
Band size should be
Amplicon 1- 3.5 kB
Amplicon 2- 3.0 kB
Sample Amplicon Pooling
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.
Pool fragments
Dilute as needed to achieve 1.0ng input concentration for library preparation
Library Prep
Follow Illumina Protocol for Nextera XT DNA Library Sample Prep
Library Pooling
Amplicon quality can effect how many samples can be pooled onto one run. Use caution in deciding how many samples to pool.
Sequencing
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.
Analysis
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
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.01516-12
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
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