Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing

Abstract

Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second-Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high-quality, full-length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene-locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD-powered CYP2D6 gene-variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy-number variation.

Keywords: CYP2D6; PacBio long-read sequencing; copy-number variation; pharmacogenomics; variant phasing.

Figure 1

Barcoding schemes. Direct versus two‐step sample barcoding. A: In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single PCR reaction. B: For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the SMRT bell library preparation.

Figure 2

Direct barcoding results. Direct barcoding results for the technical replicate with different barcodes. A: UCSC browser screenshot illustrating the detected variants (red lines) of the two fully‐phased haplogroup sequences for each sample barcode relative to the GRCh38 reference. The CYP2D6 gene is located on the negative strand. Exon numbers are indicated in white based on the NM_000106.5 transcript sequence. For the haplogroup 2 sequences, the variants determining the CYP2D6^*35 call are indicated. B: Identity and distribution of all 27 variants across the haplogroup sequences. Order in this table (top to bottom) is identical to those in Figure 2A (left to right). Variants in bold determine the CYP2D6^*35 haplotype; ^* indicates that the variant was included on the Roche AmpliChip CYP450 test; # indicates that the variant was included in PharmGKB (February 4, 2016); and “HG” and “i” denote haplogroup and index, respectively.