Analytical Validation of A 101 Germline miR-SNP Ion AmpliSeq Panel for Breast Cancer-Related Genetic Studies
DOI:
https://doi.org/10.48048/tis.2026.12823Keywords:
miRNA-SNP, Breast cancer, Ion AmpliSeq, MPS, Genotyping, ValidationAbstract
Massively parallel sequencing (MPS) technology enables simultaneous genotyping of many SNPs across multiple samples with high depth and accuracy. Such technology has become increasingly valuable for studies investigating genetic variants, including miRNA-related SNPs (miR-SNPs), that may contribute to cancer susceptibility. In this study, we designed a customized Ion AmpliSeq panel targeting 101 candidate breast cancer-associated miR-SNPs and evaluated its sequencing performance and analytical reliability on the Ion S5 XL system. Sequencing metrics, including depth of coverage (DoC), locus coverage balance (LCB), frequency of major allele reads (FMAR), and locus strand balance (LSB), were assessed across all loci. Across 50 DNA samples, the sequencing achieved a mean of 85,194 (95% CI: 79,904 - 101,503), with 91.44% (95% CI: 90.68 - 94.45) on-target reads, an average depth of 515.1× (95% CI: 488.9 - 592.9), and uniformity of 88.77% (95% CI: 88.76 - 89.64), indicating high-quality and consistent performance across libraries. Among 101 miR-SNPs, 97 loci yielded valid genotypes, while four loci resulted in no-calls due to low coverage. Genotypes obtained from the customized miR-SNP panel were fully concordant with those verified by Sanger sequencing. The panel demonstrated stable performance and high reproducibility, with consistent variant calling across replicate runs. Furthermore, reliable and complete genotyping profiles were obtained from as little as 1 ng of input DNA. Collectively, these results indicate that the customized miR-SNP MPS panel provides robust analytical performance and high accuracy, supporting its applicability for large-scale genetic and breast cancer association studies.
HIGHLIGHTS
- A 101 germline miR-SNP panel was designed using the Ion AmpliSeq platform.
- The panel targets polymorphisms in miRNA-encoding regions associated with breast cancer.
- Analytical validation demonstrated high coverage uniformity and calling accuracy.
- The panel achieved consistent performance across serial DNA dilutions and replicates.
- This validated assay supports large-scale studies of germline miR-SNPs in breast cancer risk.
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