Development and evaluation of quality metrics for bioinformatics analysis of viral insertion site data generated using high throughput sequencing

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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)195-210
Number of pages16
JournalBiomedicines
Volume2
Issue number2
DOIs
StatePublished - Jun 1 2014

Keywords

  • Integration site PCR
  • Next-generating sequencing
  • Quality metrics
  • Viral insertion site

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