Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

Summary

Insertional mutagenesis is an essential tool in forward genetics for identifying functional genomic elements. Here, we describe the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for detecting lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library.

Abstract

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens have made strides in exploring this, they often limit their scope to known genomic elements and may introduce non-specific effects. This underscores the urgent need for novel functional genomics tools that enable a deeper, unbiased understanding of genome functionality. This protocol introduces the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for identifying lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library. InSET facilitates the capture of genome-wide lentiviral integration sites, with next-generation sequencing used to detect and quantify flanking sequences. InSET's design enables the analysis of integration site abundance variations in phenotypic screens on a large scale, establishing it as a robust tool for forward genetics and for identifying functional genomic elements. A key benefit of InSET is its capacity to reveal previously unidentified genomic elements, including novel functional exons of both protein-coding and non-coding RNAs, independent of prior annotation. Overall, InSET holds significant value in studying the intricate complexity of the human genome and transcriptome, where many genomic elements await functional characterization.

Introduction

Functional genomics remains a crucial and challenging field, even in the post-genome era, with a significant portion of genomic regions still lacking functional characterization. A key reason for these challenges lies in the intricate nature of the human genome and transcriptome, which are characterized by pervasive transcription and universal alternative splicing in both protein-coding and non-coding regions^1,2,3,4,5. This complexity has been extensively highlighted through RNA-targeted enrichment studies<....

Protocol

Figure 1 provides a schematic overview of the InSET method. For optimal coverage, prepare five parallel libraries from 1 µg of genomic DNA, then pool them for next-generation sequencing. This procedure has been successfully completed continuously in previous tests, though users may introduce pause points based on their discretion. Detailed information on the reagents and equipment used in this study can be found in the Table of Materials.

Discussion

In forward genetics analysis, genome-walking is essential for profiling integration sites in insertional mutagenesis cell libraries. Classical genome-walking methods like I-PCR and LAM-PCR often require restriction enzyme digestion and ligation, which can reduce library complexity and introduce biases due to enzyme site dependency^27,28,29. The InSET method overcomes these limitations by eliminating the restriction dige.......

Materials

Name	Company	Catalog Number	Comments
0.5 M EDTA	Invitrogen	AM9260G	To make Binding/Wash Buffer
1 M Tris-HCl (Ph 8.0)	Invitrogen	15568025	To make Binding/Wash Buffer
10× Klenow Fragment Buffer	Takara	2140A
10× Taq Buffer	Tiangen	ET101-01-01
5 M NaCl	Invitrogen	AM9659	To make Binding/Wash Buffer
BeaverBeads Streptavidin	Beaver	22307-1	1 μm
BEDTools		Software, v2	https://bedtools.readthedocs.io/en/latest/
BWA-MEM			Software, v0.7.12
dNTP Mixture	Takara	4030	2.5 mM each
Equalbit dsDNA HS Assay Kit	Vazyme	EQ111-02
fastp			Software
HiSeq X Ten	Illumina	SY-412-1001	Illumina platform, outsourced to Novogene Corporation (Beijing)
Klenow Fragment	Takara	2140A
NovaSeq 6000	Illumina	20012850	Illumina platform, outsourced to Novogene Corporation (Beijing)
Qubit 3.0 Fluorometer	ThermoFisher Scientific	Q33216
SICER			Software, v1.1
Taq DNA Polymerase	Tiangen	ET101-01-01
Tween-20	HuShi	30189328	To make Binding/Wash Buffer
UltraPure Distilled Water	Invitrogen	10977015
VAHTS DNA Clean Beads	Vazyme	N411-01