1. Development of a Tagmentation-Based Next-Generation Sequencing Clinical Assay as an Alternative to Capillary Electrophoresis-Based Sequencing.
- Author
-
Kuek WCD, Chai CN, Tham WMJ, Ng AYJ, Lau DSN, Loo JYQ, Van DT, Tan JKM, Lee CK, Yan B, and Chan THM
- Subjects
- Humans, Sequence Analysis, DNA methods, Sequence Analysis, DNA standards, Polymerase Chain Reaction methods, Polymerase Chain Reaction standards, Electrophoresis, Capillary methods, High-Throughput Nucleotide Sequencing methods, High-Throughput Nucleotide Sequencing standards
- Abstract
Background: Next-generation sequencing (NGS) technology enables sample multiplexing for interrogation of multiple regions of interest (ROI). Leveraging this, together with access to affordable NGS platforms, we explored the practicality of moving capillary electrophoresis (CE), noncapillary electrophoresis and single-gene testing to NGS. In this work, we evaluated the iSeq 100's capacity to validate 89 samples at once., Methods: Genomic DNA was extracted from 89 archival samples of varying specimen types. Polymerase chain reaction (PCR) was done with in house primers, library preparation with the Nextera XT Library Preparation Kit and cleaning up with paramagnetic beads. The sequencing was performed on one Illumina iSeq 100 flow cell., Results: With our workflow, 88 out of 89 samples were accurately sequenced with variant alleles identified. One sample of the 88 samples was initially discordant because the primers used were in a heterozygous deletion region. Upon redesigning of primers, the sample proved concordant., Conclusions: The iSeq-Nextera workflow proved accurate. However, variant allele frequencys generated by the Nextera are not precise., (© 2024 The Author(s). Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)
- Published
- 2024
- Full Text
- View/download PDF