Abstract
Abstract
Background
Third-generation sequencing (TGS) enables comprehensive detection of rare hemoglobin (Hb) variants due to its long-read capability. However, library preparation is often labor-intensive and time-consuming. This study developed a novel one-step library preparation (OSLP) method for TGS. By integrating barcode sequences into target-specific primers, the approach enables immediate barcoding on amplification, streamlining identification of hemoglobinopathy variants.
Methods
A retrospective set of DNA samples (n = 455; 394 adult, 61 prenatal) underwent OSLP targeting of the HBA2/1, HBG2/1, HBD, and HBB genes on the Qitan Nanopore platform for variant and structural variation detection. Samples were divided into validation and application cohorts based on prior testing with multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing alongside Gap-polymerase chain reaction (GAP-PCR)/reverse dot blot (RDB).
Results
In the validation cohort, OSLP-TGS showed 100% concordance with routine methods (MLPA, Sanger, GAP-PCR, RDB), including 294 samples for α-globin genotyping [8 structural variants (SVs), 17 single nucleotide variants (SNVs)/insertions–deletions (indels)], 138 for β-globin genotyping (4 SVs, 22 SNVs/indels), and 61 prenatal samples for combined genotyping. The method directly spanned breakpoints and determined cis/trans configurations. In the application cohort, OSLP-TGS-Nanopore identified additional variants not covered by routine genotyping: 9 extra α-globin SNVs in 16/100 samples and 8 extra β-globin SNVs in 33/256 samples. Notably, 3 samples with the –SEA deletion harbored co-inherited novel variants, explaining their disproportionately low Hb A2 levels.
Conclusions
The OSLP-TGS-Nanopore assay provides an efficient, comprehensive solution for detecting rare hemoglobinopathy variants, with a framework extendable to population screening for diverse genetic disorders.