Benchmarking Nanopore Sequencing for CLN2 (TPP1) Mutation Detection: Integrating Rapid Genomics and Orthogonal Validation for Precision Diagnostics
Abstract
1. Introduction
2. Results
2.1. Study Population and TPP1 Enzyme Activity
2.2. Mutations in the TPP1 Gene Identified via ONT-LR Sequencing and Sanger Sequencing Validation
3. Discussion
4. Materials and Methods
4.1. Study Design and Sample Collection
4.1.1. Sample Collection
4.1.2. DBS Sample Preparation
4.1.3. Ethical Compliance and Data Collection
4.2. Biochemical Measurements
Protocols for Precision and Linearity Determination
4.3. Genomic Analysis
4.3.1. DNA Extraction
4.3.2. Primer Design and Target Amplification
4.3.3. Library Construction and Sequencing
4.3.4. Data Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AAF-AMC | Ala-Ala-Phe-7-amido-4-methylcoumarin |
ACMG | American College of Medical Genetics |
CLN2 | Ceroid Lipofuscinosis, Neuronal 2 |
DBS | Dried Blood Spot |
LP | Likely Pathogenic |
NCL | Neuronal Ceroid Lipofuscinosis |
ONT-LRS | Oxford Nanopore Technologies Long-Read Sequencing |
TPP1 | Tripeptidyl Peptidase 1 |
UCL | University College London |
VUS | Variant of Unknown Significance |
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HGVSc | HGVSp | Frequency (gnomAD/TOPMed) | Classification | Type | # Affected Alleles | RS ID |
---|---|---|---|---|---|---|
c.622C>T | p.Arg208Ter | 0.00022/0.00023 | P | Stop codon | 7 | rs119455955 |
c.1204G>T | p.Glu402Ter | 0.00000 | P | Stop codon | 4 | - |
c.857A>G | p.Asn286Ser | 0.00000/0.00001 | P | Missense codon | 2 | rs119455958 |
c.225A>G | p.Gln75 = | 0.0002/0.00006 | LP | Aberrant splicing | 1 | rs368709098 |
Primer Pair | Sequence | Tm. (°C) | Genomic Location Start-End | Fragment Length (bp) | Exons | Seq. Pools | |
---|---|---|---|---|---|---|---|
1 | TPP1_6089_F | GGCCAGTAAGTTGCAAATGTCGCACC | 67.1 | 11_6617790-6618015 | 1551 | 1-2-3 | A |
TPP1_7639_R | CCACCCTTGCCTAGCATTTGGGACC | 67.6 | 11_6619516-6619540 | ||||
2 | TPP1_4859_F | GTCCAACCACACGGGCTACTGATGC | 67.7 | 11_6616760-6616784 | 1512 | 4-5-6-7 | B |
TPP1_6370_R | TTCACAGCAGGGGGAGTGTGTGC | 67.2 | 11_6618249-6618271 | ||||
3 | TPP1_3391_F | TGGGGGCTAGAGCTCAGGAACTTCG | 67.6 | 11_6615292-6615316 | 1600 | 7-8-9-10-11 | A |
TPP1_4990_R | ACCCACGATCTCTGCTCTGACTCCC | 67.3 | 11_6616867-6616891 | ||||
4 | TPP1_2167_F | GGAGAGGGAGTGGGCAACTATGATGG | 66.2 | 11_6614068-6614093 | 1579 | 10-11-12-13 | B |
TPP1_3745_R | ACCTGGGCTATACTCACCCCTCCC | 66.8 | 11_6615623-6615646 | ||||
5 | TPP1_793_F | GCTGTAGGAGGAGGAGGAGTTTCAGC | 66.1 | 11_6612694-6612719 | 1502 | 13 | A |
TPP1_2294_R | CTGCAAGGAGACCTCTACTGTCACCG | 66.3 | 11_6614170-6614195 |
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Teker, B.; Akan, G.; Kazan, H.H.; Özgen, Ö.; Tatonyan, S.; Balci, M.C.; Karaca, M.; Kurekci, F.; Yıldız, E.P.; Güngor, O.; et al. Benchmarking Nanopore Sequencing for CLN2 (TPP1) Mutation Detection: Integrating Rapid Genomics and Orthogonal Validation for Precision Diagnostics. Int. J. Mol. Sci. 2025, 26, 5037. https://doi.org/10.3390/ijms26115037
Teker B, Akan G, Kazan HH, Özgen Ö, Tatonyan S, Balci MC, Karaca M, Kurekci F, Yıldız EP, Güngor O, et al. Benchmarking Nanopore Sequencing for CLN2 (TPP1) Mutation Detection: Integrating Rapid Genomics and Orthogonal Validation for Precision Diagnostics. International Journal of Molecular Sciences. 2025; 26(11):5037. https://doi.org/10.3390/ijms26115037
Chicago/Turabian StyleTeker, Betül, Gökce Akan, Hasan Hüseyin Kazan, Özge Özgen, Suzin Tatonyan, Mehmet Cihan Balci, Meryem Karaca, Fulya Kurekci, Edibe Pembegül Yıldız, Olcay Güngor, and et al. 2025. "Benchmarking Nanopore Sequencing for CLN2 (TPP1) Mutation Detection: Integrating Rapid Genomics and Orthogonal Validation for Precision Diagnostics" International Journal of Molecular Sciences 26, no. 11: 5037. https://doi.org/10.3390/ijms26115037
APA StyleTeker, B., Akan, G., Kazan, H. H., Özgen, Ö., Tatonyan, S., Balci, M. C., Karaca, M., Kurekci, F., Yıldız, E. P., Güngor, O., Deniz, A., Gedikbasi, A., Atalar, F., Gokcay, G. F., & Poda, M. (2025). Benchmarking Nanopore Sequencing for CLN2 (TPP1) Mutation Detection: Integrating Rapid Genomics and Orthogonal Validation for Precision Diagnostics. International Journal of Molecular Sciences, 26(11), 5037. https://doi.org/10.3390/ijms26115037