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

Introduction: Pharmacogenetics studies provide clinically relevant information on the identified associations between genetic variants and individual variability in drug response, which, in turn, offers great promise for guiding personalized drug therapy and clinical trial design. However, there is a lack of information concerning the evidence-based clinical annotations of specific genetic variants.

Aim: To design and evaluate the next-generation sequencing-based method for full-length gene polymorphism analysis.

Materials And Methods: Seven gene-specific oligonucleotide primer pairs targeting overlapping gene fragments spanning all nine gene exons with interleaving introns, untranslated (UTR) and intergenic regions were designed. Human DNA samples (n = 3) were used as a training set to check the primer performance and to optimize the PCR conditions. The effectiveness of the developed target amplification and sequencing protocol was evaluated using the test set comprising human DNA samples (n = 3) obtained from tuberculosis patients. Sequencing data analysis was performed on the Galaxy online-based platform.

Results: The sequencing data quality was sufficient for the detection of genetic variants dispersed throughout the gene with a high degree of confidence in fully covered regions achieving optimal reading depth of the targeted fragment with high base call accuracy.

Conclusion: Developed protocol can be applied in subpopulation-level association studies to determine whether single nucleotide variants (SNVs) or variant combinations from multiple regions of the gene are of clinical significance.