Next-generation sequencing-based targeted-sequencing approach for the full-length CYP3A4 gene sequencing

CYP3A4 is regarded as the most important enzyme involved in drug metabolism, and variability of CYP3A4 gene is of high pharmacogenetic importance. The next-generation sequencing (NGS) technologies enable systematic investigation of specific genomic sites or entire regions, therefore enhancing the understanding of genetic aspects that may have clinically relevant consequences. This study aimed to develop NGS-based targeted-sequencing protocol for the full-length CYP3A4 gene sequencing and apply this protocol for clinical sample analysis. Human DNA samples (n=3) were received from national biobank and used to optimize PCR and sequencing library preparation steps. Performance of developed protocol was demonstrated by analysis of tuberculosis patient DNA samples (n=5) obtained from Centre of Tuberculosis and Lung Diseases. The protocol included: full-length CYP3A4 gene amplification; amplicon pre-treatment procedure; NGS on Illumina MiSeq platform (2x250 bp paired-end reads). Sequencing data were processed on Galaxy online-based platform; detected variants were annotated using wANNOVAR tool and public databases (ClinVar, dbSNP, PharmGKB). Nine primer pairs were designed to amplify 2836-5955 bp long fragments and obtain full coverage of CYP3A4 gene. The introduction of the amplicon pre-treatment step reduced the amount non–specific amplification products. The high-quality sequencing data were generated for all clinical samples (n=5); the overall base quality was ≥35, mean read depth - 193, and sequence coverage (≥10x) 99.9%. Of the 40 single-nucleotide variants detected, 21 intronic and 1 exonic nonsynonymous variants were database-referenced, while 18 were novel and previously unreported. None of the identified variants were of clinical significance. All patients were wild-type CYP3A4*1 allele carriers (CYP3A4*1/*1 genotype). The results confirm that developed NGS-based sequencing protocol can be used to identify variable sites dispersed throughout the entire CYP3A4 gene and holds future perspective to be applied in population-level pharmacogenetic studies. Acknowledgement: This study was supported by grant No. lzp-2020/1-0050.