This work was supported the Delaney AIDS Research Enterprise (DARE) to Find a Cure (1U19AI096109 and 1UM1AI126611-01); an amfAR Research Consortium on HIV Eradication (ARCHE) Collaborative Research Grant from The Foundation for AIDS Research (amfAR 108074-50-RGRL); Australian Centre for HIV and Hepatitis Virology Research (ACH2); UCSF-GIVI Center for AIDS Research (P30 AI027763); and the Australian National Health and Medical Research Council (AAP1061681). We would like to thank Dr. Joey Lai, Genomics Facility Manager at The Westmead Institute for Medical Research for his training in library preparation and use of his facility, and the Ramaciotti Centre for Genomics (University of New South Wales, Sydney, Australia) for conducting sequencing. We acknowledge with gratitude the participants who donated samples for this study.

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+deamination+mediates+innate+immunity+to+retroviral+infection.">DNA deamination mediates innate immunity to retroviral infection.</a> Cell. 113 (6), 803-809 (2003).</li><li>Lecossier, D., Bouchonnet, F., Clavel, F., Hance, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypermutation+of+HIV-1+DNA+in+the+absence+of+the+Vif+protein.">Hypermutation of HIV-1 DNA in the absence of the Vif protein.</a> Science. 300 (5622), 1112 (2003).</li><li>Chun, T. 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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+HIV-1+evolution+in+the+gut-associated+lymphoid+tissue+from+patients+on+combination+antiviral+therapy+initiated+during+primary+infection.">Absence of HIV-1 evolution in the gut-associated lymphoid tissue from patients on combination antiviral therapy initiated during primary infection.</a> Public Library of Science Pathogens. 8 (2), e1002506 (2012).</li><li>von Stockenstrom, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+Genetic+Characterization+Reveals+That+Cell+Proliferation+Maintains+a+Persistent+HIV+Type+1+DNA+Pool+During+Effective+HIV+Therapy.">Longitudinal Genetic Characterization Reveals That Cell Proliferation Maintains a Persistent HIV Type 1 DNA Pool During Effective HIV Therapy.</a> Journal of Infectious Diseases. 212 (4), 596-607 (2015).</li><li>Palmer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple,+linked+human+immunodeficiency+virus+type+1+drug+resistance+mutations+in+treatment-experienced+patients+are+missed+by+standard+genotype+analysis.">Multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis.</a> Journal of Clinical Microbiology. 43 (1), 406-413 (2005).</li><li>. CLC Genomics Version 10 Available from: <a target="_blank" href="https://www.qiagenbioinformatics.com/products/clc-genomics-workbench">https://www.qiagenbioinformatics.com/products/clc-genomics-workbench</a> (2018)</li><li>. FastQC: a quality control tool for high throughput sequence data Available from: <a target="_blank" href="https://www.bioinformatics.babraham.ac.uk/projects/fastqc/">https://www.bioinformatics.babraham.ac.uk/projects/fastqc/</a> (2010)</li><li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30 (15), 2114-2120 (2014).</li><li>Martin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutadapt+removes+adapter+sequences+from+high-throughput+sequencing+reads.">Cutadapt removes adapter sequences from high-throughput sequencing reads.</a> EMBnet.journal. 17 (1), (2011).</li><li>Magoc, T., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLASH:+fast+length+adjustment+of+short+reads+to+improve+genome+assemblies.">FLASH: fast length adjustment of short reads to improve genome assemblies.</a> Bioinformatics. 27 (21), 2957-2963 (2011).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9 (4), 357-359 (2012).</li><li>Bankevich, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPAdes:+a+new+genome+assembly+algorithm+and+its+applications+to+single-cell+sequencing.">SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.</a> Journal of Computational Biology. 19 (5), 455-477 (2012).</li><li>Kumar, S., Stecher, G., Tamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEGA7:+Molecular+Evolutionary+Genetics+Analysis+Version+7.0+for+Bigger+Datasets.">MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets.</a> Molecular Biology and Evolution. 33 (7), 1870-1874 (2016).</li><li>Katoh, K., Standley, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFFT+multiple+sequence+alignment+software+version+7:+improvements+in+performance+and+usability.">MAFFT multiple sequence alignment software version 7: improvements in performance and usability.</a> Molecular Biology and Evolution. 30 (4), 772-780 (2013).</li><li>. HIV Sequence Database - Gene Cutter tool Available from: <a target="_blank" href="https://www.hiv.lanl.gov/content/sequence/GENE_CUTTER/cutter.html">https://www.hiv.lanl.gov/content/sequence/GENE_CUTTER/cutter.html</a> (2018)</li><li>Foster, J. L., Garcia, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Nef+in+HIV-1+replication+and+pathogenesis.">Role of Nef in HIV-1 replication and pathogenesis.</a> Advances in Pharmacology. 55, 389-409 (2007).</li><li>. HIV Sequence Database - Consensus Maker tool Available from: <a target="_blank" href="https://www.hiv.lanl.gov/content/sequence/CONSENSUS/consensus.html">https://www.hiv.lanl.gov/content/sequence/CONSENSUS/consensus.html</a> (2018)</li><li>. HIV Sequence Database - Hypermut tool Available from: <a target="_blank" href="https://www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html">https://www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html</a> (2018)</li><li>Yu, G., Smith, D. K., Zhu, H., Guan, Y., Lam, T. T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ggtree:+an+r+package+for+visualization+and+annotation+of+phylogenetic+trees+with+their+covariates+and+other+associated+data.">ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data.</a> Methods in Ecology and Evolution. , (2016).</li></ol>

The authors have nothing to disclose.

The FLIPS assay is an efficient and high-throughput method for amplifying and sequencing single, near full-length HIV-1 proviruses. Multiple factors and critical steps in the protocol that influence the number and quality of the sequences obtained have been identified. Firstly, the number of cells and the HIV-1 infection frequency of the cell population influence the number of proviruses amplified. For example, in a previous publication, approximately half as many sequences were obtained from the same number of na&#239;ve CD4+ T cells compared to effector memory CD4+ T cells. This is because na&#239;ve cells typically have a lower infection frequency than effector memory cells2. Secondly, cell lysis is preferable to column-based extraction methods for obtaining genomic DNA as there is no risk of losing DNA in the extraction process. Lastly, as with any PCR-based assay, preventing contamination is critical. Separated clean areas should be designated for preparing master mixes, handling genomic DNA, adding positive controls, DNA purification and quantification, and library preparation. This is particularly important for single-copy assays such as the one presented here.
Implementation of the FLIPS assay should first include running a positive control such as pNL4-3 plasmids rather than participant samples. This will allow for any troubleshooting prior to the use of HIV-1 positive cells, as the sequences obtained can be compared to available reference sequences for these plasmids. When using HIV-1 positive cells, it is important to consider the HIV-1 subtype (primers designed for FLIPS are specific to subtype B) and the infection frequency of the cell population if little to no proviruses are amplified. Primer sequences can be modified/redesigned to match other subtypes. Additionally, a well containing a positive control should be included in every PCR performed.
FLIPS has overcome the limitations of previous sequencing assays, including SPS. Through amplifying and sequencing near full-length HIV-1 proviruses, FLIPS can determine the potential replication-competency of HIV-1 proviruses. This was not possible using SPS, which sequenced only sub-genomic regions and therefore selected for sequences with intact primer binding sites. Furthermore, FLIPS overcomes the limitations associated with utilizing multiple amplification and sequencing primers, as was employed by previous full-length sequencing assays1,4. Through two rounds of PCR targeting the HIV-1 LTR regions combined with NGS, FLIPS decreases the number and complexity of primers required. FLIPS is therefore less susceptible to the consequences of primer mismatches, namely the erroneous identification of defective proviruses and an inability to amplify some proviruses within a population. The FLIPS protocol is also more efficient and allows for a higher throughput of sequencing than previous methods.
Evidently, FLIPS provides advantages over existing methods that determine the genetic composition of HIV-1 proviruses. However, it is important to acknowledge limitations of FLIPS. Firstly, the FLIPS assay has not been developed as a tool for measuring the size of the latent HIV-1 reservoir, as analyses to determine whether FLIPS amplifies every HIV-1 provirus present in a cell population have not been completed. FLIPS is instead useful for making relative comparisons of the composition of the reservoir between different cell populations2. Secondly, the replication-competency of intact HIV-1 proviruses cannot be determined with certainty without in vivo analyses, such as those performed by Ho et al.4. Thirdly, FLIPS is not designed to determine the integration site of HIV-1 proviruses.
Minor variations to the FLIPS protocol can increase its application. For example, changes in primer sequences can allow different and multiple HIV-1 subtypes to be amplified and sequenced. Sequencing of plasma HIV-1 virions is possible through the addition of cDNA synthesis prior to nested PCR. Future utilization of single molecule sequencing methods will eliminate the need for de novo assembly.
Genetic sequencing of integrated HIV-1 proviruses has increased our understanding of the latent HIV-1 reservoir. FLIPS is an important tool for future studies elucidating the composition and distribution of the latent reservoir. However, the application of FLIPS can extend beyond the reservoir. Future studies may utilize FLIPS to determine particular targets for CRISPR-Cas technology, or assist in the identification of coding and non-coding regions which make the virus more responsive to latency reversing agents. Viral recombination may be better understood by looking at the junction sites of large internal deletions.

Genetic characterization of the latent HIV-1 reservoir, which persists in individuals on long-term antiretroviral therapy (ART), has been vital to understanding that the majority of integrated proviruses are defective and replication-incompetent1,2. During the process of reverse transcription, errors are introduced into the integrated proviral sequence. Some mechanisms that generate defective proviral sequences include the error-prone HIV-1 reverse transcriptase enzyme3, template switching4, and/or APOBEC-induced hypermutation5,6. Two recent studies have found that approximately 5% of HIV-1 proviruses isolated from individuals on long-term ART are genetically intact, and potentially replication-competent, and may contribute to the rapid rebound in HIV-1 plasma levels upon cessation of ART1,2,7. Previous studies have identified that replication-competent HIV-1 proviruses persist in na&#239;ve and resting memory CD4+ T cell subsets (including central, transitional and effector memory T cells), indicating the importance of targeting these cells in future eradication strategies2,8,9.
Early insights into the distribution, dynamics and maintenance of the latent HIV-1 reservoir were achieved through utilization of single-proviral sequencing (SPS) methods that genetically characterize sub-genomic regions of the HIV-1 genome10,11,12,13. SPS is a versatile tool, able to sequence a single HIV-1 provirus from within a single infected cell. However, SPS is unable to determine the replication-competency of proviruses, since it only sequences sub-genomic regions and misses proviruses that contain large deletions within primer binding sites. A previous study has demonstrated that SPS overestimates the size of the replication-competent reservoir by 13- to 17-fold through selectively sequencing intact sub-genomic regions2.
To address the limitations of SPS, Ho et al.4 and Bruner et al.1 developed assays to sequence near full-length HIV-1 proviruses. This allowed the frequency of genetically intact, and potentially replication-competent, HIV-1 proviruses in individuals on long-term ART to be determined. These assays amplified and sequenced (via Sanger sequencing) sub-genomic regions that were then assembled to obtain a sequence of the (intact or defective) HIV-1 provirus. Three limitations of this approach are: 1) the use of multiple sequencing primers increases the risk of unintentionally introducing defects into the proviral sequence, 2) primer mismatches may prevent amplification of particular proviruses, and 3) often the entire proviral sequence cannot be resolved due to the technicality of these methods.
To overcome the limitations of existing full-length HIV-1 proviral sequencing assays, we developed the Full-Length Individual Proviral Sequencing (FLIPS) assay. FLIPS is a next-generation sequencing (NGS)-based assay which amplifies and sequences near full-length (intact or defective) HIV-1 proviruses in a high-throughput and efficient manner. FLIPS provides advantages over previous assays, as it limits the number of primers utilized; therefore, it decreases the chance of primer mismatches, which may limit the population of proviruses captured or unintentionally introduce defects into a viral sequence. FLIPS is also less technically challenging than previous assays and involves 6 main steps: 1) lysis of HIV-1 infected cells, 2) amplification of single HIV-1 proviruses via nested PCR performed at limiting dilution using primers specific for the highly conserved HIV-1 5&#39; and 3&#39; U5 LTR region (Figure 1A), 3) purification and quantification of amplified products, 4) library preparation of amplified proviruses for NGS, 5) NGS, and 6) de novo assembly of sequenced proviruses to obtain contigs of each individual provirus.
Sequences generated by FLIPS can undergo a stringent process of elimination to identify those which are genetically intact and potentially replication-competent (Figure 1C)2. Genetically intact proviruses lack all known defects which result in generation of a replication-incompetent provirus. These defects include: inversion sequences, large internal deletions, hypermutation/deleterious stop codons, frameshifts, or mutations in the 5&#39; packaging signal or major splice donor (MSD) site.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58016/58016fig1.jpg" /> 
Figure 1: Critical steps in the full-length individual proviral sequencing (FLIPS) assay. (A) HIV-1 DNA genome with primer binding sites in 5&#39; and 3&#39; U5 LTR regions used by FLIPS to amplify near full-length (defective and intact) HIV-1 proviruses via nested PCR. (B) Layout of a 96-well PCR plate containing 80 sample wells (20 wells for each dilution), 4 negative control wells, and 1 positive control well. (C) Process of elimination used to identify genetically intact, and potentially replication-competent, HIV-1 proviruses. This figure has been modified from Hiener et al.2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58016/58016fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>UltraPure 1 M Tris-HCI, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15568025</td>
			<td>Dilute to 5 mM for nested PCR</td>
		</tr>
		<tr>
			<td>Nonidet P 40 Substitute solution</td>
			<td>Sigma</td>
			<td>98379</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K Solution (20 mg/mL)</td>
			<td>Promega</td>
			<td>AM2548</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well thermocycler</td>
			<td></td>
			<td></td>
			<td>Any 96 well thermocycler can be used</td>
		</tr>
		<tr>
			<td>Platinum Taq DNA Polymerase High Fidelity</td>
			<td>Invitrogen</td>
			<td>11304011</td>
			<td></td>
		</tr>
		<tr>
			<td>10X High Fidelity Buffer [600 mM Tris-SO4 (pH 8.9), 180 mM (NH4)2SO4]</td>
			<td>Invitrogen</td>
			<td>11304011</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mM MgSO4</td>
			<td>Invitrogen</td>
			<td>11304011</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Nucleotide Mix, 10 mM</td>
			<td>Promega</td>
			<td>C1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure H2O</td>
			<td>Invitrogen</td>
			<td>10977023</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR1 and PCR2 Primers</td>
			<td></td>
			<td></td>
			<td>Desalted. Dilute to (100 M) with H2O</td>
		</tr>
		<tr>
			<td>PCR Plate 96 Well, Half Skirt, Single Notch Corner, Clear</td>
			<td>Axygen</td>
			<td>PCR-96M2-HS-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; Adhesive Seals</td>
			<td>BioRad</td>
			<td>MSB1001</td>
			<td>Required to seal 96 well PCR plates</td>
		</tr>
		<tr>
			<td>HIV-1 NL4-3 Infectious Molecular Clone (pNL4-3)</td>
			<td>The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 NL4-3 Infectious Molecular Clone (pNL4-3) from Dr. Malcolm Martin (Cat# 114).</td>
			<td></td>
			<td>Diluted to 10^5 copies/mL and used as positive control</td>
		</tr>
		<tr>
			<td>PCR plate spinner or benchtop centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E-GEL 48 1% Agarose</td>
			<td>Invitrogen</td>
			<td>G800801</td>
			<td>Precast 1% agarose gels (two gels used per 96 well plate). Any 1% agarose gel can be substituted. Contains ethidium bromide.</td>
		</tr>
		<tr>
			<td>DirectLoad Wide Range DNA Marker</td>
			<td>Sigma</td>
			<td>D7058</td>
			<td>Any ladder with a range up to 10 kb can be substituted</td>
		</tr>
		<tr>
			<td>Mother E-Base device</td>
			<td>Invitrogen</td>
			<td>EBM03</td>
			<td>Required for running precast 48 well 1% agarose gels</td>
		</tr>
		<tr>
			<td>Gel Doc EZ Gel Documentation System</td>
			<td>BioRad</td>
			<td>1708270</td>
			<td>Any visualisation system for ethidium bromide containing agarose gels can be used</td>
		</tr>
		<tr>
			<td>PlateMax Peelable Heat Sealing</td>
			<td>Axygen</td>
			<td>HS-200</td>
			<td>Heat sealing film for long term storage</td>
		</tr>
		<tr>
			<td>96 Well 0.8 mL Storage Plate</td>
			<td>ThermoFisher Scientific</td>
			<td>AB0765</td>
			<td>0.8 mL 96 well plate required for magnetic bead based PCR purification</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP (PCR purification kit)</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td>Magnetic bead based PCR purification kit. Other PCR purification kits can be substitued here (e.g. QIAquick PCR Purification Kit (Qiagen Cat#28106)</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure. 200 proof, for molecular biology</td>
			<td>Sigma</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stand-96</td>
			<td>Invitrogen</td>
			<td>AM10027</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate shaker</td>
			<td></td>
			<td></td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Buffer EB</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td>Elution buffer</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Assay Kit</td>
			<td>Invitrogen</td>
			<td>P11496</td>
			<td>A fluorescence based stain for measuring dsDNA concentration</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT DNA Library Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-131-1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT Index Kit</td>
			<td>Illumina</td>
			<td>FC-131-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hard-Shell 96-Well PCR Plates</td>
			<td>Biorad</td>
			<td>HSP9601</td>
			<td>Required for Nextera XT DNA library preparation kit</td>
		</tr>
		<tr>
			<td>Library Quantification Kit - Illumina/Universal</td>
			<td>Kapa Biosystems</td>
			<td>KK4824</td>
			<td>Other library quantification kits can be substitued (e.g. JetSeq DNA Library Quantification Lo-Rox Kit (Bioline Cat#BIO-68029)</td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer</td>
			<td>Agilent Technology</td>
			<td></td>
			<td>Automated electrophoresis system . Use in conjunction with a High Sensistivity DNA kit</td>
		</tr>
		<tr>
			<td>High Sensistivty DNA kit</td>
			<td>Agilent Technology</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina MiSeq Platform</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

amplification of near full-length hiv-1 proviruses for next-generation sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

The FLIPS assay amplifies and sequences single, near full-length HIV-1 proviruses. The protocol involves 6 steps to obtain near full-length proviral sequences. These steps include: lysis of infected cells, nested PCR of full-length (intact and defective) HIV-1 proviruses, DNA purification and quantification, sequencing library preparation, NGS, and de novo assembly of sequenced proviruses. The end of each step can be considered a checkpoint in which the quality of the product (e.g. amplified DNA, purified DNA, sequencing library or sequence) can be assessed prior to the next step. An overview of the assessment performed at the end of each step and the expected results is outlined below.
Following nested PCR, amplified products are run on a 1% agarose gel (Figure 3). The initial quality of the PCR can be determined by inspection of negative and positive controls. Negative control wells containing amplified product indicate contamination and positive control wells absent of amplified product indicate insufficient amplification. Next, wells containing amplified product are selected for sequencing. To avoid wells containing mixtures of multiple amplified proviruses, only wells containing amplified product run at end-point dilution are considered for sequencing. According to Poisson distribution, the end-point dilution is found when 30% of wells are positive for amplified product. At this dilution, there is an 80% chance these wells contain a single amplified provirus. Additionally, wells containing multiple amplified proviruses of different lengths can be visualized at this stage as multiple bands will appear on the gel. These wells should not be selected for sequencing (Figure 3).
Following purification of the amplified proviruses selected for sequencing, quantification ensures no proviral DNA is lost during the purification stage. If the DNA concentration of an amplified provirus is &#60;0.2 ng/&#181;L, the remaining sample in the PCR2 plate can be purified. A similar checkpoint occurs following library preparation, in which each individual library is quantified. This ensures individual proviruses have been appropriately fragmented, tagged and amplified prior to sequencing. Individual proviral libraries are pooled in equimolar amounts to a final library concentration of 4 nM (or as specified by the sequencing provider). If the concentration of an individual proviral library is too low, it can be excluded from the sequencing library pool, or the individual library prepared again. A final check of the concentration of the pooled library is performed prior to sequencing along with confirming the average fragment lengths.
Quality control steps before the de novo assembly stage ensures the quality of the reads used to assemble the final proviral contig. These steps include: removal of adapter sequences, trimming of 5&#39; and 3&#39; nucleotides, a stringent quality limit, and disregarding short reads. CLC Genomics Workbench can provide quality control reports that can be used before to assess the initial quality of the reads and guide trimming settings, and then after to determine if the trimming was sufficient to remove low quality regions. Additionally, for de novo assembly, the quality of the assembled contigs can be assessed for sufficient depth (&#62;1000X) and evenness of coverage (Figure 4A). Mixed populations can also be identified at this stage. Mixtures of multiple full-length (~9 kb) proviruses are identified through the presence of multiple SNPs with a frequency of greater than 40%, whereas mixtures of short (containing a large internal deletion) and full-length proviruses can be identified by uneven read coverage following mapping to a full-length reference from the same participant (Figure 4B).
Depending on the application, the final alignment can be visualized using tools such as ggtree available as a package in &#34;R: A language and environment for statistical computing&#34;27. In a recent study, FLIPS was utilized to sequence HIV-1 proviruses from na&#239;ve, central, transitional, and effector memory CD4+ T cells isolated from individuals on long-term ART, with the aim to identify if particular cell subsets showed higher proportions of genetically intact and potentially replication-competent HIV-12. Here, visual representation of the sequences isolated from one participant of this study (participant 2026) is presented (Figure 5). In this participant, the majority (97%) of sequences were defective, with intact sequences found in effector and transitional memory CD4+ T cells. This visualization tool is useful for showing the number of sequences with large internal deletions and their position in the genome. It can be annotated further to indicate sequences with deleterious stop codons, frameshift mutations, and deletions/mutations in the MSD site and/or packaging signal.
One application of the FLIPS assay is the identification of genetically intact and potentially replication-competent HIV-1 proviruses. In a recent study of 531 sequences isolated from CD4+ T cells from 6 participants on long-term ART, 26 (5%) genetically intact HIV-1 proviruses were identified2. The remaining defective proviruses included those with inversion sequences (6%), large internal deletions (68%), deleterious stop codons/hypermutation (9%), frameshift mutations (1%), and defects in the packaging signal and/or mutations in the MSD site (11%).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58016/58016fig2.jpg" /> 
Figure 2: Overview of workflow for de novo assembly of HIV-1 proviruses. The major steps in the workflow include: 1) sequence read quality control, 2) merging overlapping pairs, 3) de novo assembly, and 4) remapping and consensus building have been colored red, blue, green and orange, respectively. This figure has been modified from Hiener et al.2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58016/58016fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58016/58016fig3.jpg" /> 
Figure 3: Example agarose gel of PCR amplified HIV-1 proviruses. Wells 1, 2, 3, 6, 9 and 10 contain amplified HIV-1 proviruses. Well 10 contains a provirus with a large internal deletion, well 2 contains co-amplification of two HIV-1 proviruses of different lengths (mixture), and well 12 contains positive control. Note the percent of wells containing amplified product is 60%, which is above the percentage required to isolate single templates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58016/58016fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58016/58016fig4.jpg" /> 
Figure 4: Example output of read mapping. (A) Example demonstrating even coverage due to the amplification of a single full-length HIV-1 provirus. Following de novo assembly, all reads are mapped to the assembled contig to produce a consensus sequence. The software platform allows the mapped reads to be inspected for sufficient and even coverage. (B) Example demonstrating co-amplification of two HIV-1 proviruses of different lengths (mixture). To determine mixtures, reads are mapped to a full-length (~9 kb) reference sequence from the same participant and read mapping inspected. The presence of uneven coverage indicates a mixture. This figure is reproduced with permission from Qiagen14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58016/58016fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58016/58016fig5.jpg" /> 
Figure 5: Example visualization of HIV-1 proviral sequences isolated from CD4+ T cell subsets from an individual on long-term ART. Individual HIV-1 proviral sequences are represented by horizontal lines. This figure has been modified from Hiener et al.2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58016/58016fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing at JoVE.com

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

amplification-near-full-length-hiv-1-proviruses-for-next-generation

Nanyang Technological University

Research

JoVE Journal

Genetics

11.9K Views.  University of Sydney. Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

Video: Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the traditional method of choice is 3&#39; rapid amplification of cDNA ends (3&#39; RACE). Protocols for 3&#39; RACE require the careful design and selection of nested primers within the 3&#39; untranslated region (3&#39; UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3&#39; UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3&#39; UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3&#39; RACE. Expanded 3&#39; RACE can detect unusual 3&#39; UTRs, including gene fusions within the 3&#39; UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

The two different 3&#39; rapid amplification of cDNA ends (3&#39; RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3&#39; UTR of a transcript using RNA obtained from different cancer cell lines.

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...