Genome walking by next generation sequencing approaches.

Mariateresa Volpicella,Immacolata Fanizza,Alessandra Costanza,Luigi R Ceci,Antonio Placido,Claudia Leoni

doi:10.3390/biology1030495

Abstract

Genome Walking (GW) comprises a number of PCR-based methods for the identification of nucleotide sequences flanking known regions. The different methods have been used for several purposes: from de novo sequencing, useful for the identification of unknown regions, to the characterization of insertion sites for viruses and transposons. In the latter cases Genome Walking methods have been recently boosted by coupling to Next Generation Sequencing technologies. This review will focus on the development of several protocols for the application of Next Generation Sequencing (NGS) technologies to GW, which have been developed in the course of analysis of insertional libraries. These analyses find broad application in protocols for functional genomics and gene therapy. Thanks to the application of NGS technologies, the original vision of GW as a procedure for walking along an unknown genome is now changing into the possibility of observing the parallel marching of hundreds of thousands of primers across the borders of inserted DNA molecules in host genomes.

Highlights

The identification of unknown nucleotide sequences starting from a previously identified DNA region can be directly obtained by a number of Genome Walking (GW) methods all having in common a final PCR amplification in which an oligonucleotide specific for the known sequence is coupled with an oligonucleotide derived from the adopted GW strategy
GW is a highly flexible approach, allowing both the identification of specific, unique sequences and the analysis of large libraries
In this review we focus on the application of Next Generation Sequencing (NGS) to GW

Summary

Introduction

The identification of unknown nucleotide sequences starting from a previously identified DNA region can be directly obtained by a number of Genome Walking (GW) methods all having in common a final PCR amplification in which an oligonucleotide specific for the known sequence is coupled with an oligonucleotide derived from the adopted GW strategy. GW is a highly flexible approach, allowing both the identification of specific, unique sequences (as for the analysis of single gene flanking sequences) and the analysis of large libraries (such as those obtained by insertional mutagenesis of retroviruses and transposable elements). In the latter case, GW has the potential to be powered by the enormous capacity of Generation Sequencing (NGS). A first report on the application of pyrosequencing (using the Roche 454 platform) to GW is that of Wang et al [6], while the first application of SBS methods (using the Illumina technology) to GW is more recent [7]. A final paragraph gives some general considerations about the possibility to use other, as yet unexplored, combinations of NGS and GW methods

GW by 454-Pyrosequencing

GW by Illumina-SBS

Findings

Other Possible NGS-GW Pairing and General Conclusions