Applying the National Genomic DNA Reference Materials to Evaluate the Performance of Nanopore Sequencing in Identifying Thalassemia Variants

Abstract

Objectives: Nanopore sequencing shows advantages in detecting single nucleotide variations (SNVs), deletions, and complex structural variants as a single test in thalassemia. However, the performance evaluation or verification of this method remains unestablished, which is essential before clinical utility and panel registration. Here, we developed a classification method for thalassemia mutations, enabling automated interpretation, visual representation, and identification of diverse mutation types.

Methods: We used a total of 36 samples, comprising 32 reference materials and four clinical samples to assess the performance of nanopore sequencing in identifying variants in terms of concordance, precision, and the lower limits of detection.

Results: Our analysis successfully identified 19 SNVs, six deletions, and two triplications using nanopore sequencing across all samples. Notably, these variants showed complete concordance of 100% with the genotypes of the reference materials and known results. The precision of nanopore sequencing for detecting thalassemia variants was consistently high, with neither false positive nor false negative observed. Furthermore, the lower limits of detection achieved in our study were 3 ng/μL.

Conclusions: Overall, our study proved that the reference materials can be used to evaluate the performance of nanopore sequencing in identifying thalassemia mutations, and it is necessary to incorporate triplications when utilizing reference materials for performance evaluation of long-read sequencing. The consistent and robust performance of nanopore sequencing in this study demonstrates its potential as a reliable method for comprehensive variant detection in thalassemia and other genetic diseases diagnosis.

Keywords: Long read sequencing; nanopore sequencing; national reference material; thalassemia; variant.

FIGURE 1

The selection diagram and amplicons for performance evaluation. (A) The selected diagram of samples for performance evaluation. (B) Primer positions and target amplicon ranges of multiplex PCR for α−/β‐globin genes.

FIGURE 2

Interpretation of SVs of α‐globin gene. The blue band means sequencing depth of the target amplicons within the α‐globin gene. A, B, C, D and E represent the amplicons for HBA2, HBA1, HBA, SEA, and THAI, respectively. If A to E exhibit the sequencing depth as in “Nor”, then the sample is a normal genotype. If A, B, D and E present the “Del”, and C present “Del3.7” or “Del4.2”, then we could identify the sample as −α^3.7 or −α^4.2. Similarly, If A, B present as “Del”, C present as “Nor”, while D or E present as “Del”, then we could interpret the sample as ‐‐^SEA or ‐‐^THAI deletions. If A, B, D and E present the “Del”, and C present “Dup3.7” or “Dup4.2”, then we could identify the sample ααα^anti3.7 or ααα^anti4.2 triplications. Nor, normal; Del, deletion; Dup, duplication.

FIGURE 3

Interpretation of SVs of β‐globin gene. The blue bands signify sequencing depth of the target amplicons within the β‐globin genes. A, B, and C correspond to the amplicons of SEA‐HPFH, HBDB, and ^Gγ(^Aγδβ)⁰, respectively. If A, B, and C present “Nor”, then we could interpret the sample as normal genotypes. If B present “Del”, and A or C present “Del”, then we could identify the sample as SEA‐HPFH or ^Gγ(^Aγδβ)⁰ deletions. Nor, normal; Del, deletion.