Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Summary

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Abstract

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

Introduction

Retroviruses insert their genomic DNA into the host genome at various sites. This unique property, which may contribute to the development of cancers and other forms of viral pathogenesis, has the ironic benefit of making these viruses highly amenable to cellular engineering for gene therapy and basic biology research. The viral Integration Site (IS) – the location on the host genome where a foreign DNA (virus) is integrated – has important implications for the fate of both the integrated viruses and the host cells. IS assays have been used in various biological and clinical research settings to study retroviral integration site selection and pathogenesis,....

Protocol

1. Generating Upstream (left)- and Downstream (right)-junction Sequence Libraries

1. DNA linker preparation:
   1. Prepare a 10 µL linker DNA solution by adding 2 µL of 100 µM LINKER_A oligos (final: 20 µM), 2 µL of 100 µM LINKER_B oligos (final: 20 µM), 2 µL of 5 M NaCl (final: 1M), and 4 µL of nuclease-free water in a PCR tube. See Table 1 for the linker sequences.
   2. Incubate the linker DNA solution at 95 °C for 5 min in a PCR instrument, stop the run program, and turn the PCR instrument power off. Leave the linker solution in the instrument for 30 min to slow....

Results

The bidirectional IS assay generated different sizes of PCR amplicons for both the upstream (left) and downstream (right) vector host junctions (Figure 2). The size of a PCR amplicon is dependent on the location of the nearest GTAC motif upstream and downstream from an integrome. The assay also produced internal DNA PCR amplicons: retroviral sequences near the polypurine tract and the primer binding site were concomitantly amplified during left- and right-jun.......

Discussion

The bidirectional assay enables the simultaneous analysis of both the upstream (left) and downstream (right) vector-host DNA junction sequences and is useful in a number of gene therapy, stem cell, and cancer research applications. The use of GTAC-motif enzymes (RsaI and CviQI) and the bidirectional PCR approach significantly improves the chances of detecting an integrome (or a clonal population) when compared to previous TCGA-motif enzyme (TaqαI)-based assays^2,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Thermostable DNA polymerase	Agilent	600424	PicoMaxx Polymerase
Thermostable DNA polymerase buffer	Agilent	600424	PicoMaxx Polymerase buffer
Deoxynucleotide (dNTP) solution mix	New England Biolabs	N0447L	dNTP solution mix (10 mM each)
PCR tubes	VWR International	53509-304	PCR Strip Tubes With Individual Attached Caps
2 mL microcentrifuge tube	Molecular Bioproducts	3453	microcentrifuge tubes
PCR purification kit	Qiagen	28106
RsaI	New England Biolabs	R0167L	restriction enzyme
CviQI	New England Biolabs	R0639L	restriction enzyme
Buffer A	New England Biolabs	B7204S	NEB CutSmart buffer
DNA Polymerase I large (klenow) fragment	New England Biolabs	M0210L	Blunting
streptavidin beads solution	Invitrogen	60101	Dynabeads kilobaseBINDER kit
Binding Solution	Invitrogen	60101	Dynabeads kilobaseBINDER kit
Washing Solution	Invitrogen	60101	Dynabeads kilobaseBINDER kit
magnetic stand	ThermoFisher	12321D	DynaMag™-2 Magnet
T4 DNA ligase	New England Biolabs	M0202L	T4 DNA ligase
10X T4 DNA ligase buffer	New England Biolabs	B0202S	T4 DNA ligase reaction buffer
5X T4 DNA ligase buffer	Invitrogen	46300-018	T4 DNA ligase buffer with polyethylene glycol-8000
UV-Vis spectrophotometer	Fisher Scientific	S06497	Nanodrop 2000
pvuII	new England Biolabs	R0151L	restriction enzyme
sfoI	new England Biolabs	R0606L	restriction enzyme
Buffer B	new England Biolabs	B7203S	NEB buffer 3.1
Nuclease free water	Integrated DNA Technologies	11-05-01-14
Capillary electrophoresis	Qiagen	9001941	QIAxcel capillary electrophoresis
Veriti 96-well Fast Thermal Cycler	Thermo Fisher Scientific	4375305	PCR Instrument
Rotating wheel (or Roller)	Eppendorf	M10534004	Cell Culture Roller Drums
DNA size marker	Qiagen	929559	QX size marker (100 - 2,500 bp)
DNA size marker	Qiagen	929554	QX size marker (50 - 1,500 bp)
DNA alignment markers	Qiagen	929524	QX DNA Alignment Marker
genomc DNA	Not Available	Not Available	Sample genomc DNA from in vivo or in vitro experiments