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Validation of a semiconductor next-generation sequencing assay for the clinical genetic screening of CFTR.

Trujillano D, Weiss ME, Köster J, Papachristos EB, Werber M, Kandaswamy KK, Marais A, Eichler S, Creed J, Baysal E, Jaber IY, Mehaney DA, Farra C, Rolfs A - Mol Genet Genomic Med (2015)

Bottom Line: In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases.Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly-thymidine-guanine and poly-thymidine (poly-TG-T) tracts were also detected.Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene.

View Article: PubMed Central - PubMed

Affiliation: Centogene AG Rostock, Germany.

ABSTRACT
Genetic testing for cystic fibrosis and CFTR-related disorders mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the CFTR gene. We have explored a more efficient genetic screening strategy based on next-generation sequencing (NGS) of the CFTR gene. We validated this approach in a cohort of 177 patients with previously known CFTR mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq™ CFTR panel. The DNA libraries were pooled, barcoded, and sequenced using an Ion Torrent PGM sequencer. The combination of different robust bioinformatics tools allowed us to detect previously known pathogenic mutations and polymorphisms in the 177 samples, without detecting spurious pathogenic calls. In summary, the assay achieves a sensitivity of 94.45% (95% CI: 92% to 96.9%), with a specificity of detecting nonvariant sites from the CFTR reference sequence of 100% (95% CI: 100% to 100%), a positive predictive value of 100% (95% CI: 100% to 100%), and a negative predictive value of 99.99% (95% CI: 99.99% to 100%). In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases. Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly-thymidine-guanine and poly-thymidine (poly-TG-T) tracts were also detected. Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene.

No MeSH data available.


Related in: MedlinePlus

Representation of the average depth of coverage of CFTR across the 177 patients. Green lines represent the exons of the gene. Red lines represent the amplicons of the assay.
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fig01: Representation of the average depth of coverage of CFTR across the 177 patients. Green lines represent the exons of the gene. Red lines represent the amplicons of the assay.

Mentions: From these data, we can conclude that all samples were uniformly covered at depths that in all cases exceed by far the minimum coverage required for reliable variant calling (Fig.1). The minor differences between samples were neutralized by the excessive overall coverage achieved by the assay. The sequence quality metrics of this data warrant a confident detection of variants in all patients.


Validation of a semiconductor next-generation sequencing assay for the clinical genetic screening of CFTR.

Trujillano D, Weiss ME, Köster J, Papachristos EB, Werber M, Kandaswamy KK, Marais A, Eichler S, Creed J, Baysal E, Jaber IY, Mehaney DA, Farra C, Rolfs A - Mol Genet Genomic Med (2015)

Representation of the average depth of coverage of CFTR across the 177 patients. Green lines represent the exons of the gene. Red lines represent the amplicons of the assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585447&req=5

fig01: Representation of the average depth of coverage of CFTR across the 177 patients. Green lines represent the exons of the gene. Red lines represent the amplicons of the assay.
Mentions: From these data, we can conclude that all samples were uniformly covered at depths that in all cases exceed by far the minimum coverage required for reliable variant calling (Fig.1). The minor differences between samples were neutralized by the excessive overall coverage achieved by the assay. The sequence quality metrics of this data warrant a confident detection of variants in all patients.

Bottom Line: In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases.Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly-thymidine-guanine and poly-thymidine (poly-TG-T) tracts were also detected.Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene.

View Article: PubMed Central - PubMed

Affiliation: Centogene AG Rostock, Germany.

ABSTRACT
Genetic testing for cystic fibrosis and CFTR-related disorders mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the CFTR gene. We have explored a more efficient genetic screening strategy based on next-generation sequencing (NGS) of the CFTR gene. We validated this approach in a cohort of 177 patients with previously known CFTR mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq™ CFTR panel. The DNA libraries were pooled, barcoded, and sequenced using an Ion Torrent PGM sequencer. The combination of different robust bioinformatics tools allowed us to detect previously known pathogenic mutations and polymorphisms in the 177 samples, without detecting spurious pathogenic calls. In summary, the assay achieves a sensitivity of 94.45% (95% CI: 92% to 96.9%), with a specificity of detecting nonvariant sites from the CFTR reference sequence of 100% (95% CI: 100% to 100%), a positive predictive value of 100% (95% CI: 100% to 100%), and a negative predictive value of 99.99% (95% CI: 99.99% to 100%). In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases. Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly-thymidine-guanine and poly-thymidine (poly-TG-T) tracts were also detected. Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene.

No MeSH data available.


Related in: MedlinePlus