Name: amplification of near full-length hiv-1 proviruses for next-generation sequencing
Uploaded: 2018-10-16T13:00:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing at JoVE.com

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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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-SO&lt;sub&gt;4&lt;/sub&gt; (pH 8.9), 180 mM (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;]</td><td>Invitrogen</td><td>11304011</td><td></td></tr><tr><td>50 mM MgSO&lt;sub&gt;4&lt;/sub&gt;</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 H&lt;sub&gt;2&lt;/sub&gt;O</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 &amp;micro;M) with H&lt;sub&gt;2&lt;/sub&gt;O</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 &#039;B&#039; 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&lt;sup&gt;5&lt;/sup&gt; 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 (&lt;em&gt;e.g.&lt;/em&gt; 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 (&lt;em&gt;e.g.&lt;/em&gt; 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 ...

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Nanyang Technological University

Research

JoVE Journal

Genetics

12.0K Views.  University of Sydney. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van HIV, zoals het bepalen van de genetische samenstelling van HIV-provirussen en verschillende celtypen. Vooral het identificeren van degenen die genetisch intact en potentieel replicatie competent. De belangrijkste voordelen van deze techniek is dat het ons in staat stelt om bijna volledige lengte HIV provirussen uit enkele HIV geïnfecteerde cellen te versterken en deze vervolgens te sequencen door de volgend...

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

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Immunology and Infection

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

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 ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

Here we present a next-generation in situ hybridization assay to identify specific viral RNA or DNA sequences in formalin-fixed paraffin embedded (FFPE) tissues. This approach allows visualization of low copies of RNA and DNA in less than 24 h with very high sensitivity and specificity.

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important ...

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Here, we describe the method used to determine the required read coverage, using the comparison between the R10 flow cell and Illumina sequencing as an example.

We previously validated a protocol for amplicon-based whole genome Usutu virus (USUV) sequencing on a nanopore sequencing platform. Here, we describe the methods used in more detail and determine the error rate of the nanopore R10 flow cell.

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for ...

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

Medicine

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

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

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