January 6, 2016  |  General

Scientists Develop More Accurate Variant-Calling Procedure for Important Drug Metabolism Enzyme Using SMRT Sequencing

In an article entitled “Long-read single-molecule real-time (SMRT) full gene sequencing of cytochrome P450-2D6 (CYP2D6)” in Human Mutation, authors Wangiong Qiao, Yao Yang, Stuart Scott and other colleagues at the Icahn School of Medicine at Mount Sinai demonstrate a new way of analyzing the CYP2D6 gene using PacBio long reads. This gene has been shown to have a central role in drug metabolism and is believed to be directly involved in the metabolism of ~25% of all commonly used drugs. Given its importance, CYP2D6 genotype testing is now being widely used to predict how efficiently patients will metabolize drugs such as codeine, antidepressants, or antipsychotics.
Studying CYP2D6 presents many challenges. It is highly polymorphic, “with over 100 variant star (*) alleles catalogued, many of which are associated with reduced or no enzyme activity,” the authors report. In addition, it is highly prone to copy number variation. Both gene duplications and deletions can occur, with pseudogenes maintaining high sequence homology to functional copies. As a result, “Accurate prediction of CYP2D6 metabolizer status necessitates direct analysis of the duplicated gene copy (or copies) when an increased copy number is detected, particularly when identified concurrently with normal activity and loss-of-function alleles in compound heterozygosity,” the authors write. Such copy number changes can alter the interpretation of CYP2D6 phenotypes.
In their publication, the authors describe targeted long-read sequencing of the CYP2D6 gene along with upstream and downstream gene copies by using targeted long-range PCR and barcoding for multiplexing. The analysis consisted of demultiplexing, read alignment using BWA-MEM software, and error correction using Amplicon Long-read Error Correction (ALEC) that was developed by the authors. The team began by validating their SMRT Sequencing pipeline with 10 previously characterized DNA samples, showing that not only were they able to correctly call genotypes, but their approach provided additional information about variants that had been missed by other platforms. Specifically, SMRT Sequencing enabled the team to further refine the genotypes, reclassify diplotypes (two haplotypes, i.e., multiple genotypes on homologous chromosomes), characterize allele-specific duplication, and discover novel alleles.
After validation work, they then applied their method to 14 samples that previously had been found to yield inconclusive or unreliable results. SMRT Sequencing was able to reconcile the discrepancies that had been seen from the other platforms and provide new data. “In addition to confirming consensus diplotypes, CYP2D6 SMRT sequencing enabled suballele resolution, genotype refinement, duplicated allele characterization, and discovery of a novel tandem arrangement,” the scientists report.
The authors conclude, “Long-read CYP2D6 SMRT sequencing is an innovative, reproducible, and validated method for full-gene characterization, duplication allele-specific analysis and novel allele discovery, which will likely improve CYP2D6 metabolizer phenotype prediction for both research and clinical testing applications.”
You can also watch Dr. Stuart Scott describing this research in a presentation he gave at the American Society of Human Genetics meeting in 2014.

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