Simultaneous detection of thalassemia, hemoglobinopathies, and G6PD variants using long-read nanopore sequencing: genetic complexity and heterogeneity in Thailand.

Multiplex long-amplicon PCR followed by nanopore sequencing, variant calling with Clair3 and Ensembl VEP annotation, covering HBA1, HBA2, HBB and G6PD on a single platform.

Thalassemia, hemoglobinopathies and G6PD deficiency are highly prevalent and genetically heterogeneous in Thailand, and conventional molecular diagnosis requires multiple targeted assays to cover deletional and non-deletional variants. The authors developed and analytically validated a single long-read workflow on Oxford Nanopore technology detecting pathogenic variants in HBA1, HBA2, HBB and G6PD simultaneously, using multiplex long-amplicon PCR, Clair3 variant calling, Ensembl VEP annotation and ACMG classification. Sixty-three genomic DNA samples with previously characterized genotypes were analyzed, all successfully genotyped with 100% concordance with conventional PCR and Sanger sequencing. Thirty-nine distinct variants were detected and classified, including large alpha-globin deletions, beta-thalassemia mutations, structural hemoglobin variants and the major Southeast Asian G6PD variants. Long-read sequencing resolved the homology between HBA1 and HBA2 and characterized multilocus co-inheritance patterns.

Synthesis written by Geno'X. For the full original abstract, please refer to the source publication.

The gain here is organizational rather than analytical: replacing a cascade of targeted assays with a single run cuts turnaround time and cost, which matters in a population where these disorders are common and genetic counseling depends on the couple. The caveat is the study design: 63 previously genotyped samples measure concordance, not prospective yield or the rate of unresolved genotypes in unselected referrals. As with any amplicon-based approach, coverage is bounded by the chosen primers — a deletion extending beyond the amplicons would be missed, which must be stated explicitly before routine use.

Analysis by Dr Thibaut Benquey