Name: amplification, next-generation sequencing, and genomic dna mapping of retroviral integration sites
Uploaded: 2016-03-22T15:00:00
Description: Watch this Scientific Journal Video about Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites at JoVE.com

We are grateful to our colleagues Stephen Hughes and Henry Levin for advice that was critical to establish the NGS protocol for retroviral integration site sequencing in the Engelman lab. This work was supported by US National Institutes of Health grants AI039394 and AI052014 (to A.N.E.) and AI060354 (Harvard University Center for AIDS Research).

1. Generate Virus Stocks
Note: A flow chart of the wet bench aspect of this protocol is depicted in Figure 1. The details of viral stock production and subsequent infection of tissue culture cells will generally apply to different types of retroviruses. For some experiments, the target cell may not express the endogenous viral receptor(s), and in such cases the construction of pseudotyped retroviral particles harboring heterologous viral envelope glycoprotein, e.g. the ...

A protocol for the analysis of retroviral integration sites, from the initial virus infection step through mapping of genomic distribution patterns, is described. This protocol is applicable to any retrovirus and any infectable cell type. Furthermore, the assay pipeline is quite sensitive, with the potential to recover a satisfactory number of unique integration sites from serial dilutions of genomic DNA equivalent to that of an infection initiated with an MOI of 6.4 x 10-5. This sensitivity makes the protocol...

Integration of viral DNA (vDNA) into the host cell genome is an essential step in the retroviral life cycle. Integration is accomplished by the viral enzyme integrase (IN), which carries out two distinct catalytic processes that lead to the establishment of the stably inserted provirus 1. IN subunits engage the ends of the linear vDNA that is generated through reverse transcription, forming the higher-order intasome with vDNA ends held together by an IN multimer 2-4. IN cleaves the 3&#39; ends of the vDNA downstream from invariant 5&#39;-CA-3&#39; sequences in a process known as 3&#39;-processing, leaving recessed 3&#39; ends with reactive hydroxyl groups at each vDNA terminus 5-8. The intasome is subsequently imported into the nucleus as part of a large assembly of host and viral proteins known as the preintegration complex (PIC) 9-11. After encountering cellular target DNA (tDNA), IN uses the vDNA 3&#39;-hydroxyl groups to cleave the tDNA top and bottom strands in a staggered fashion and simultaneously joins the vDNA to tDNA 5&#39; phosphate groups through the process of strand transfer 12,13.
Retroviruses exhibit integration site preferences on the local and global scales. Locally, consensus integration sites consist of weakly conserved palindromic tDNA sequences that span from approximately five to ten bp upstream and downstream from the vDNA insertion sites 14,15. Globally, retroviruses target specific chromatin annotations 16. There are seven different retroviral genera - alpha through epsilon, lenti, and spuma. The lentiviruses, which include HIV-1, favor integration within the bodies of actively transcribed genes 17, while the gammaretroviruses preferentially integrate into transcriptional start sites (TSSs) and active enhancer regions 18-20. In sharp contrast, spumavirus is strongly biased towards heterochromatic regions, such as gene-poor lamina-associated domains 21. Local tDNA base preferences are in large part dictated by specific networks of nucleoprotein contacts between IN and tDNA 13,22,23. For the lentiviruses and gammaretroviruses, integration relative to genomic annotations is in large part governed by interactions between IN and cognate cellular factors 24-27. Altering the specifics of the IN-tDNA interaction network 13,22,23,28 and disrupting or re-engineering IN-host factor interactions 25-27,29-32 are proven strategies to retarget integration on the local and global levels, respectively.
The power of DNA sequencing procedures used to catalogue retroviral integration sites has increased immensely over the past decades. Integration sites were recovered in pioneering work using laborious purification and manual cloning techniques to yield just a handful of unique sites per study 33,34. The combination of LM-PCR amplification of LTR-host DNA junctions with the ability to map individual integration sites to human and mouse draft genomes transformed the field, with the number of sites recovered from exogenous tissue culture cell infections increasing to several hundred to thousands 17,18. The more recent combination of LM-PCR with NGS methodology has sent library depth skyrocketing. Specifically, pyrosequencing yielded on the order of tens of thousands of unique integration sites 30,35-38, while libraries sequenced through the use of DNA clustering can yield millions of unique sequences 19-21,39. Here we describe an optimized LM-PCR protocol for amplifying and sequencing retroviral integration sites using DNA clustering NGS. The method incorporates required adapter sequences into the PCR primers and hence directly into the amplified DNA molecules, thereby precluding the requirement for an additional adapter ligation step prior to sequencing 40. The bioinformatic analysis pipeline, from the parsing of raw sequencing data for LTR-host DNA junctions to the mapping of unique integration sites to pertinent genomic features, is also generally described. In accordance with the precedence established from prior methodological protocols in this field 36,38,41-43, custom scripts can be developed to aid the completion of specific steps in the bioinformatics pipeline. The utility and sensitivity of the protocol is illustrated with representative data by amplifying, sequencing, and mapping HIV-1 integration sites from tissue culture cells infected at the approximate multiplicity of infection (MOI) of 1.0, as well as a titration series of this DNA diluted through uninfected cellular DNA in 5-fold steps to a maximum dilution of 1:15,625 to yield the approximate equivalent MOI of 6.4 x 10-5.

<table border="0" cellpadding="0" cellspacing="0" width="1326">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">DMEM</td>
			<td style="width:199px;">Gibco</td>
			<td style="width:111px;">11965-084</td>
			<td style="width:780px;">Standard cell culture medium, compatible with HEK293T cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Fetal Bovine Serum</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:111px;">SH 30088.03</td>
			<td>Different lots of serum may need to be pre-screened for optimal viral production</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Penicillin/Streptomycin</td>
			<td style="width:199px;">Corning</td>
			<td style="width:111px;">30-002-Cl</td>
			<td>Antibiotics to be added to DMEM</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Phosphate-buffered saline</td>
			<td style="width:199px;">Mediatech</td>
			<td style="width:111px;">21-040-CV</td>
			<td>Used to wash cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Trypsin EDTA</td>
			<td style="width:199px;">Corning</td>
			<td style="width:111px;">25-053-CI</td>
			<td>Used to detach adherent cells from tissue culture plates</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">PolyJet</td>
			<td style="width:199px;">SignaGen Laboratories</td>
			<td style="width:111px;">SL100688</td>
			<td>DNA transfection reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:237px;">0.45 &#181;m Filters</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td>09-740-35B</td>
			<td>Used to filter virus particle-containing cell culture media</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Turbo DNase</td>
			<td style="width:199px;">Ambion</td>
			<td style="width:111px;">AM2239</td>
			<td>Used to degrade carryover plasmid DNA from virus stocks</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">HIV-1 p24 Antigen Capture Assay</td>
			<td style="width:199px;">ABL Inc.</td>
			<td style="width:111px;">5447</td>
			<td>Used to quantify yield of virus production</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">DNeasy Blood &#38; Tissue Kit</td>
			<td style="width:199px;">Qiagen</td>
			<td style="width:111px;">69506</td>
			<td>Used to purify genomic DNA from cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Sonicator</td>
			<td style="width:199px;">Covaris</td>
			<td style="width:111px;">S2</td>
			<td>With this model of sonicator perform two rounds of duty cycle, 5%; intensity, 3; cycles per burst, 200; time, 80 sec</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Nuclease-Free Water</td>
			<td style="width:199px;">GeneMate</td>
			<td style="width:111px;">G-3250-125</td>
			<td>Commercially-available water is recommended to reduce the possibility of sample cross-contamination</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">QIAQuick PCR Purification Kit</td>
			<td style="width:199px;">Qiagen</td>
			<td style="width:111px;">28106</td>
			<td>Used to purify DNA during library construction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">End-It DNA End-Repair Kit</td>
			<td style="width:199px;">Epicentre</td>
			<td style="width:111px;">ER81050</td>
			<td>Used to repair DNA ends of sonicated DNA samples</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Klenow Fragment (3&#39;-5&#39; exo&#8211;)</td>
			<td style="width:199px;">New England Biolabs (NEB)</td>
			<td style="width:111px;">M0212S</td>
			<td>Used with dATP to A-tail repaired DNA fragments</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">dATP</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:111px;">R0141</td>
			<td>Deoxyadenosine triphosphate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">MseI</td>
			<td style="width:199px;">NEB</td>
			<td>R0525L</td>
			<td>Restriction endonuclease for genomic DNA cleavage</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">BglII</td>
			<td style="width:199px;">NEB</td>
			<td style="width:111px;">R0144L</td>
			<td>Restriction endonuclease to suppress amplification of upstream HIV-1 U5 sequence</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">T4 DNA Ligase</td>
			<td style="width:199px;">NEB</td>
			<td style="width:111px;">M0202L/6218</td>
			<td>Enzyme for covalent joining of compatible DNA ends</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DNA Oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td>custom</td>
			<td>Have the company purify the oligos. HPLC purification suffices for DNAs &#60;30 nucleotides; PAGE purify longer DNAs&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Advantage 2 Polymerase Mix</td>
			<td style="width:199px;">Clontech</td>
			<td style="width:111px;">639202</td>
			<td>Commercial mix containing DNA polymerase for PCR</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">dNTPs (100 mM solutions)</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:111px;">R0181</td>
			<td>Dilute the four chemicals on ice with sterile water to reach the intermediate worrking concentrations of 2.5 mM each dNTP</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">NanoDrop</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:111px;">NanoDrop 2000</td>
			<td>Spectrophotometer for determination of DNA concentration</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">Qubit Fluorimeter</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:111px;">Qubit&#174; 3.0</td>
			<td>Fluorometer used to confirm integration site library DNA concentration</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">2200 TapeStation System</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:111px;">G2964AA</td>
			<td>Tape-based assay to confirm integration site library DNA size distribution</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:237px;">MiSeq</td>
			<td style="width:199px;">Illumina</td>
			<td style="width:111px;">SY-410-1003</td>
			<td>Used for NGS</td>
		</tr>
	</tbody>
</table>

amplification, next-generation sequencing, and genomic dna mapping of retroviral integration sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

Table 4 lists the results of a representative experiment to illustrate the sensitivity of NGS for recovering integration sites from a culture of infected cells. Uninfected cellular DNA was utilized to serially dilute genomic DNA from an infection in which every cell on average contained one integration 40. Dilutions were prepared in steps of five to a maximal dilution of 1:15,625. Genomic DNA in the titration series was then fragmented by sonication or by diges...

Watch this Scientific Journal Video about Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites at JoVE.com

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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