Development and Evaluation of Quality Metrics for Bioinformatics Analysis of Viral Insertion Site Data Generated Using High Throughput Sequencing.

Hongyu Gao,Mary Dinauer,Keithanne Mockaitis,Yu-Hsiang Chen,Aparna Jasti,Kenneth Cornetta,Troy Hawkins

doi:10.3390/biomedicines2020195

Abstract

Integration of viral vectors into a host genome is associated with insertional mutagenesis and subjects in clinical gene therapy trials must be monitored for this adverse event. Several PCR based methods such as ligase-mediated (LM) PCR, linear-amplification-mediated (LAM) PCR and non-restrictive (nr) LAM PCR were developed to identify sites of vector integration. Coupling the power of next-generation sequencing technologies with various PCR approaches will provide a comprehensive and genome-wide profiling of insertion sites and increase throughput. In this bioinformatics study, we aimed to develop and apply quality metrics to viral insertion data obtained using next-generation sequencing. We developed five simple metrics for assessing next-generation sequencing data from different PCR products and showed how the metrics can be used to objectively compare runs performed with the same methodology as well as data generated using different PCR techniques. The results will help researchers troubleshoot complex methodologies, understand the quality of sequencing data, and provide a starting point for developing standardization of vector insertion site data analysis.

Highlights

Gene therapy, using retroviral (RV) and lentiviral (LV) vectors, holds great promise for treatment of a wide variety of genetic disorders and diseases
In this study we developed a series of quality metrics and applied these to sequence data obtained using generation sequencing technology
The theoretical yield (TY) is a readout of the number of reads you expect might contain useful data for a single LM-PCR sample sequenced in high throughput: TY = Nl>n/NT, where l is read length and n is a threshold length that is defined by the length of the Long Terminal Repeat (LTR) after the final round primer position +

Summary

Introduction

Gene therapy, using retroviral (RV) and lentiviral (LV) vectors, holds great promise for treatment of a wide variety of genetic disorders and diseases These integrating vectors, have the risk of insertional mutagenesis and cause unintended consequences when integration occurs in or near host genes involved in regulating cell growth and division [1,2,3,4]. Ligase-mediated (LM) PCR [5,6,7,8], linear-amplification-mediated (LAM) PCR [9,10,11], and most recently non-restrictive (nr) LAM PCR [12,13] select and amplify regions of genomic DNA immediately flanking terminal vector sequences. They have been used as biomarkers for tracking the growth and distribution of individual clones during repopulation [14]

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