Implementation of a Next-Generation Sequencing-Based Targeted Approach for Full-Length CYP3A4 Gene Sequencing

Abstract

Aim: To evaluate the application of next-generation sequencing-based targeted protocol for full-length CYP3A4 gene sequencing analysis. Materials & methods: The developed sequencing protocol was applied to analyze human DNA samples (n = 7) obtained from tuberculosis patients admitted to the Riga East University Hospital, Center of Tuberculosis and Lung diseases. Results: The sequencing data quality was sufficient for the detection of already known genetic variants, as well as for identifying rare and novel variants dispersed throughout the CYP3A4 gene with a high degree of confidence. Conclusion: Developed protocol can be applied in subpopulation level association studies to determine whether specific genetic variants or variant combinations from multiple regions of the CYP3A4 gene are of clinical significance.

Next-Generation Sequencing and Bioinformatics-Based Protocol for the Full-Length CYP2E1 Gene Polymorphism Analysis

“…PCR amplification of large-sized gene fragments, NGS library preparation workflow and subsequent data analysis was carried out according to the protocol by Kivrane et al with a few minor modifications. 12 Briefly, a total of 20–35 ng of DNA per reaction was used; the PCR reaction was performed using All Taq PCR Core Kit (QIAGEN, Germany) following the manufacturer’s protocol for large-sized fragment amplification; the PCR amplicon size ranged from 2059 to 3820 bp ( Table 1 ). Obtained PCR products were analyzed by 1.5% agarose gel electrophoresis.…”