Open AccessGenotyping of familial Mediterranean fever gene (MEFV)—Single nucleotide polymorphism—Comparison of Nanopore with conventional Sanger sequencing
Author(s) -
Jonas Schmidt,
Sandro Berghaus,
Frithjof Blessing,
Holger Herbeck,
J. Blessing,
Peter Schierack,
Stefan Rödiger,
Dirk Roggenbuck,
Folker Wenzel
Publication year - 2022
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0265622
Subject(s) - genotyping , sanger sequencing , familial mediterranean fever , mefv , genetics , single nucleotide polymorphism , nanopore sequencing , biology , genotype , polymorphism (computer science) , exon , gene , dna sequencing , medicine , gene mutation , mutation , disease , pathology
Background Through continuous innovation and improvement, Nanopore sequencing has become a powerful technology. Because of its fast processing time, low cost, and ability to generate long reads, this sequencing technique would be particularly suitable for clinical diagnostics. However, its raw data accuracy is inferior in contrast to other sequencing technologies. This constraint still results in limited use of Nanopore sequencing in the field of clinical diagnostics and requires further validation and IVD certification. Methods We evaluated the performance of latest Nanopore sequencing in combination with a dedicated data-analysis pipeline for single nucleotide polymorphism (SNP) genotyping of the familial Mediterranean fever gene ( MEFV ) by amplicon sequencing of 47 clinical samples. Mutations in MEFV are associated with Mediterranean fever, a hereditary periodic fever syndrome. Conventional Sanger sequencing, which is commonly applied in clinical genetic diagnostics, was used as a reference method. Results Nanopore sequencing enabled the sequencing of 10 target regions within MEFV with high read depth (median read depth 7565x) in all samples and identified a total of 435 SNPs in the whole sample collective, of which 29 were unique. Comparison of both sequencing workflows showed a near perfect agreement with no false negative calls. Precision, Recall, and F1-Score of the Nanopore sequencing workflow were > 0.99, respectively. Conclusions These results demonstrated the great potential of current Nanopore sequencing for application in clinical diagnostics, at least for SNP genotyping by amplicon sequencing. Other more complex applications, especially structural variant identification, require further in-depth clinical validation.