Development of a Tagmentation-Based Next-Generation Sequencing Clinical Assay As an Alternative to Capillary Electrophoresis-Based Sequencing

Overview

Journal Mol Genet Genomic Med

Specialty Genetics

Date 2024 Nov 19

PMID 39560203

Authors

Wei Cheng David Kuek

Chean Nee Chai

Wei Ming Jason Tham

Alvin Yu Jin Ng

Dilys Shi Ning Lau

Janice Yen Qi Loo

Dan Thu Van

Joanna Kia Min Tan

Chun Kiat Lee

Benedict Yan

Tim Hon Man Chan

Affiliations

Soon will be listed here.

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.

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