Sep 17, 2024
Version 1
FL-BEEHIV HIV-1 Genotyping and Drug Resistance by Next Generation Sequencing-High throughput extraction V.1
- 1Florida Department of Health;
- 2Florida Department of Health, Bureau of Public Health Laboratories
- HIV
Protocol Citation: Brenna M McGruder Rawson, Jason Blanton 2024. FL-BEEHIV HIV-1 Genotyping and Drug Resistance by Next Generation Sequencing-High throughput extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8x3ov5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 17, 2024
Last Modified: September 17, 2024
Protocol Integer ID: 107759
Keywords: HIV-1, HIV-1 genotyping, HIV-1 drug resistance, Next Generation sequencing, drug resistance mutations
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Abstract
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.
Materials
RNA Extraction by Qiagen Viral RNA Mini Kit (DSP or Regular)
SSIV VILO Master Mix (Thermofisher, Cat 11756050)
Q5 Master Mix (NEB, Cat M0492S)
Primers (Gall A, et al. Journal of Clinical Microbiology. 2012; 50:12)
Set and primer Sequence (5’–3’) Positionsa Product sizea
2
Pan-HIV-1_2F GGG AAG TGA YAT AGC WGG AAC 1031–1051 3,574 bp
Pan-HIV-1_2R CTG CCA TCT GTT TTC CAT ART C 4604–4583
3
Pan-HIV-1_3F TTA AAA GAA AAG GGG GGA TTG GG 4329–4351 3,066 bp
Pan-HIV-1_3R TGG CYT GTA CCG TCA GCG 7394–7377
aAccording to HIV-1 reference strain HXB2 (GenBank accession number NC001802).
Single/multichannel pipettes with p20/p200/p1000 tips
Thermocycler
Nuclease-free water
AMPure XP Beads (Beckman Coulter)
Magnetic stand
Tapestation or Agarose gel
Qubit or other quantitation method
Illumina Nextera XT DNA Library Prep Kit
Illumina Nextera v2 Index Kits
Illumina iSeq
Illumina iSeq 100 i1 v2 cartridge
Pipeline: HIV1-PR+RT+IN (2.2.0HIV1 V1.6)
RNA Extraction
RNA Extraction
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.
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
Run the program on the KingFisher Flex (200uL)
cDNA Synthesis
cDNA Synthesis
25m
25m
Master Mix
4.0 µL SuperScript IV VILO Master Mix
6.0 µL Nuclease Free Water
10.0 µL RNA template
Run the following protocol on a thermocyler
25 °C 00:10:00
50 °C 00:10:00
85 °C 00:05:00
25m
Amplicon PCR
Amplicon PCR
6m
6m
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
12.5 µL 2x Q5 Master Mix
0.5 µL Forward Primer 20 micromolar (µM)
0.5 µL Reverse Primer 20 micromolar (µM)
6.5 µL Nuclease Free Water
5.0 µL cDNA template
Run the following protocol on a thermocycler
105 °C Lid
50 °C 00:00:30
98 °C 00:00:30
35 cycles
72 °C 00:05:00
4 °C Hold
6m
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
Fragment Normalizing and Pooling
Fragment Normalizing and Pooling
Fragments can be pooled in eqimolar amounts or in equal concentrations.
Pool fragments
Dilute as needed to achieve 1.0 ng input concentration for library preparation
Library Prep
Library Prep
Follow Illumina Protocol for Nextera XT DNA Library Sample Prep
Pooling Samples
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
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
References
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