Use of the QIAGEN GeneReader NGS system for detection of KRAS mutations, validated by the QIAGEN Therascreen PCR kit and alternative NGS platform.

BACKGROUND: The detection of somatic mutations in primary tumors is critical for the understanding of cancer evolution and targeting therapy. Multiple technologies have been developed to enable the detection of such mutations. Next generation sequencing (NGS) is a new platform that is gradually becoming the technology of choice for genotyping cancer samples, owing to its ability to simultaneously interrogate many genomic loci at massively high efficiency and increasingly lower cost. However, multiple barriers still exist for its broader adoption in clinical research practice, such as fragmented workflow and complex bioinformatics analysis and interpretation. METHODS: We performed validation of the QIAGEN GeneReader NGS System using the QIAact Actionable Insights Tumor Panel, focusing on clinically meaningful mutations by using DNA extracted from formalin-fixed paraffin-embedded (FFPE) colorectal tissue with known KRAS mutations. The performance of the GeneReader was evaluated and compared to data generated from alternative technologies (PCR and pyrosequencing) as well as an alternative NGS platform. The results were further confirmed with Sanger sequencing. RESULTS: The data generated from the GeneReader achieved 100% concordance with reference technologies. Furthermore, the GeneReader workflow provides a truly integrated workflow, eliminating artifacts resulting from routine sample preparation; and providing up-to-date interpretation of test results. CONCLUSION: The GeneReader NGS system offers an effective and efficient method to identify somatic (KRAS) cancer mutations.

Target silencing of components of the conserved oligomeric Golgi complex impairs HIV-1 replication.

All viruses require host cell factors to replicate. A large number of host factors have been identified that participate at numerous points of the human immunodeficiency virus 1 (HIV-1) life cycle. Recent evidence supports a role for components of the trans-Golgi network (TGN) in mediating early steps in the HIV-1 life cycle. The conserved oligomeric Golgi (COG) complex is a heteroctamer complex that functions in coat protein complex I (COPI)-mediated intra-Golgi retrograde trafficking and plays an important role in the maintenance of Golgi structure and integrity as well as glycosylation enzyme homeostasis. The targeted silencing of components of lobe B of the COG complex, namely COG5, COG6, COG7 and COG8, inhibited HIV-1 replication. This inhibition of HIV-1 replication preceded late reverse transcription (RT) but did not affect viral fusion. Silencing of the COG interacting protein the t-SNARE syntaxin 5, showed a similar defect in late RT product formation, strengthening the role of the TGN in HIV replication.