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Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping

Kaat Rubben (UGent) , Laurentijn Tilleman (UGent) , Koen Deserranno (UGent) , Olivier Tytgat, Dieter Deforce (UGent) and Filip Van Nieuwerburgh (UGent)
(2022) PLOS GENETICS. 18(9).
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Abstract
Abstract: CYP2D6 is a very important pharmacogene as it is responsible for the metabolization or bioactivation of 20 to 30% of the clinically used drugs. However, despite its relatively small length of only 4.4 kb, it is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants.
Keywords
Cancer Research, Genetics (clinical), Genetics, Molecular Biology, Ecology, Evolution, Behavior and Systematics, CYP2D6, CYTOCHROME-P450, GENE

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Citation

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MLA
Rubben, Kaat, et al. “Cas9 Targeted Nanopore Sequencing with Enhanced Variant Calling Improves CYP2D6-CYP2D7 Hybrid Allele Genotyping.” PLOS GENETICS, edited by Guanzheng Luo, vol. 18, no. 9, 2022, doi:10.1371/journal.pgen.1010176.
APA
Rubben, K., Tilleman, L., Deserranno, K., Tytgat, O., Deforce, D., & Van Nieuwerburgh, F. (2022). Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping. PLOS GENETICS, 18(9). https://doi.org/10.1371/journal.pgen.1010176
Chicago author-date
Rubben, Kaat, Laurentijn Tilleman, Koen Deserranno, Olivier Tytgat, Dieter Deforce, and Filip Van Nieuwerburgh. 2022. “Cas9 Targeted Nanopore Sequencing with Enhanced Variant Calling Improves CYP2D6-CYP2D7 Hybrid Allele Genotyping.” Edited by Guanzheng Luo. PLOS GENETICS 18 (9). https://doi.org/10.1371/journal.pgen.1010176.
Chicago author-date (all authors)
Rubben, Kaat, Laurentijn Tilleman, Koen Deserranno, Olivier Tytgat, Dieter Deforce, and Filip Van Nieuwerburgh. 2022. “Cas9 Targeted Nanopore Sequencing with Enhanced Variant Calling Improves CYP2D6-CYP2D7 Hybrid Allele Genotyping.” Ed by. Guanzheng Luo. PLOS GENETICS 18 (9). doi:10.1371/journal.pgen.1010176.
Vancouver
1.
Rubben K, Tilleman L, Deserranno K, Tytgat O, Deforce D, Van Nieuwerburgh F. Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping. Luo G, editor. PLOS GENETICS. 2022;18(9).
IEEE
[1]
K. Rubben, L. Tilleman, K. Deserranno, O. Tytgat, D. Deforce, and F. Van Nieuwerburgh, “Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping,” PLOS GENETICS, vol. 18, no. 9, 2022.
@article{8771189,
  abstract     = {{Abstract: CYP2D6 is a very important pharmacogene as it is responsible for the metabolization or bioactivation of 20 to 30% of the clinically used drugs. However, despite its relatively small length of only 4.4 kb, it is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants.}},
  articleno    = {{e1010176}},
  author       = {{Rubben, Kaat and Tilleman, Laurentijn and Deserranno, Koen and Tytgat, Olivier and Deforce, Dieter and Van Nieuwerburgh, Filip}},
  editor       = {{Luo, Guanzheng}},
  issn         = {{1553-7404}},
  journal      = {{PLOS GENETICS}},
  keywords     = {{Cancer Research,Genetics (clinical),Genetics,Molecular Biology,Ecology,Evolution,Behavior and Systematics,CYP2D6,CYTOCHROME-P450,GENE}},
  language     = {{eng}},
  number       = {{9}},
  pages        = {{21}},
  title        = {{Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping}},
  url          = {{http://doi.org/10.1371/journal.pgen.1010176}},
  volume       = {{18}},
  year         = {{2022}},
}
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