Oxford Nanopore Technology (ONT) Sequencing provides a cost-effective solution for long-read sequencing with minimal laboratory setup. MES also requires less input DNA than WGS does. The effectiveness of exome sequencing in variant detection of rare autosomal recessive monogenic disorder and diseases of high genetic heterogeneity have been well examined. MES uses customized or commercially available capture panels that target a subset of the entire exome, covering genes and positions with clinical significance. Thus, target enrichment sequencing of a subset of the genome, using medical exome sequencing (MES), for example, has become a common alternative solution. However, obtaining sufficient depth of coverage (DoC) for accurate variant calling with whole genome sequencing (WGS) is still costly for routine clinical tests. High-throughput sequencing allows efficient and comprehensive screening of all clinically significant genomic positions for patients with potential genetic defects. Screening of single genes with traditional techniques such as Sanger sequencing is tedious and time-consuming, especially in heterogeneous diseases, where variants in different genes can result in similar phenotypes. The long-read exon captured data has potential for further development, promoting the application of long-read sequencing in personalized disease treatment and risk prediction. The workflow is cost-effective, with a short turnaround time for high accuracy variant calling in 4800 clinically significant genes and regions using a single MinION flowcell. We presented ECNano, an out-of-the-box workflow comprising (1) a wet-lab protocol for ONT target enrichment sequencing and (2) a downstream variant detection workflow, Clair-ensemble. The whole workflow from wet-lab protocol to variant detection was completed within three days. Clair-ensemble achieved > 99% recall and accuracy for SNV calling. The long reads of ECNano also covered the adjacent splice sites well, with 98.5% of positions having ≥ 30× DoC. ECNano obtained an average read length of 1000 bp. For accurate ONT variant calling, the generated reads sufficiently covered 98.9% of pathogenic positions listed in ClinVar, with 98.96% having at least 30× DoC. ResultsĮCNano achieved deep on-target depth of coverage (DoC) at average > 100× and > 98% uniformity using one MinION flowcell. To evaluate its performance and practicality, ECNano was tested on both reference DNA samples and patient samples. The subsequent variant-calling workflow, Clair-ensemble, adopted a fast RNN-based variant caller, Clair, and was optimized for target enrichment data. The ECNano wet-lab protocol was optimized to perform long-read target enrichment and ONT library preparation to stably generate high-quality MES data with adequate coverage. We introduced a cost-effective optimized workflow, ECNano, comprising a wet-lab protocol and bioinformatics analysis, for accurate variant detection at 4800 clinically important genes and regions using a single MinION flowcell. By applying MES with MinION sequencing, the technology can achieve a more uniform capture of the target regions, shorter turnaround time, and lower sequencing cost per sample. Medical exome sequencing (MES) targets clinically significant exon regions, allowing rapid and comprehensive screening of pathogenic variants. Having a high sequencing error of ONT and limited throughput from a single MinION flowcell, however, limits its applicability for accurate variant detection. The application of long-read sequencing using the Oxford Nanopore Technologies (ONT) MinION sequencer is getting more diverse in the medical field.
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