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

Biomedicines

Keithanne Mockaitis¹  Yu-Hsiang Chen²  Hongyu Gao³  Troy Hawkins³  Aparna Jasti³  Kenneth Cornetta³  Mary Dinauer⁴ 

[1] Department of Biology and Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405-3700, USA;Department of Biology, Indiana University–Purdue University, Indianapolis, IN 46202, USA;Department of Medical and Molecular Genetics, Indiana University School of Medicine, IB 130, 975 West Walnut Street, Indianapolis, IN 46202, USA;Department of Pediatrics and Pathology and Immunology, Washington University, St. Louis,MO 63110, USA;

关键词: viral insertion site;    quality metrics;    next-generating sequencing;    integration site PCR;   

DOI  :  10.3390/biomedicines2020195

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown