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

Summary

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.

Abstract

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.

Introduction

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' ends of the vDNA downstream from invariant 5'-CA-3' sequences in a process known as 3'-processing, leaving recessed 3' ends with reactive hydrox....

Protocol

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 G glycoprotein from vesicular stomatitis virus (VSV-G), will be required for infection ^44,45.

Note: Precaution ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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