Noninvasive prenatal testing for \u03b2-thalassemia by targeted nanopore sequencing combined with relative haplotype dosage (RHDO): a feasibility study

Yuhuan Xie,Yang Wang,Yufan Li,Chunsheng Zhang,Yulai Guo,JiaJia Xian,BoLe Du,Shuming Ouyang,Fuman Jiang,Sheng Li,Xiaofang Sun,Yinghong Yang,Min Chen,Xiaojing Zeng,Longmei Zhang,Weiqiang Liu,Fei Luo

doi:10.1038/s41598-021-85128-2

Abstract

Noninvasive prenatal testing (NIPT) for single gene disorders remains challenging. One approach that allows for accurate detection of the slight increase of the maternally inherited allele is the relative haplotype dosage (RHDO) analysis, which requires the construction of parental haplotypes. Recently, the nanopore sequencing technologies have become available and may be an ideal tool for direct construction of haplotypes. Here, we explored the feasibility of combining nanopore sequencing with the RHDO analysis in NIPT of β-thalassemia. Thirteen families at risk for β-thalassemia were recruited. Targeted region of parental genomic DNA was amplified by long-range PCR of 10 kb and 20 kb amplicons. Parental haplotypes were constructed using nanopore sequencing and next generation sequencing data. Fetal inheritance of parental haplotypes was classified by the RHDO analysis using data from maternal plasma DNA sequencing. Haplotype phasing was achieved in 12 families using data from 10 kb library. While data from the 20 kb library gave a better performance that haplotype phasing was achieved in all 13 families. Fetal status was correctly classified in 12 out of 13 families. Thus, targeted nanopore sequencing combined with the RHDO analysis is feasible to NIPT for β-thalassemia.

Highlights

The application of noninvasive prenatal testing (NIPT) for single gene disorders (SGDs) remains challenging
We explored the feasibility of applying nanopore sequencing technology combined with the relative haplotype dosage (RHDO) analysis in Noninvasive prenatal testing (NIPT) for β-thalassemia, an autosomal recessive blood disease, enabling to build an accurate, cost-effective and convenient method for prenatal testing for SGDs
Inheritance of parental haplotypes was classified by the RHDO analysis through the maternal plasma DNA sequencing data

Summary

Introduction

The application of noninvasive prenatal testing (NIPT) for single gene disorders (SGDs) remains challenging. Compared with RMD approach, RHDO analyzes a range of SNP allele counts within a haplotype, which undoubtedly makes the statistical test more robust[5] This strategy was successfully applied in several types of SGD studies, such as spinal muscular a trophy[8], Duchenne and Becker muscular d ystrophies[9], β-thalassemia[5,10], congenital adrenal h yperplasia[11,12], Ellis-van Creveld syndrome, hemophilia and Hunter s yndrome[13] and so on. We explored the feasibility of applying nanopore sequencing technology combined with the RHDO analysis in NIPT for β-thalassemia, an autosomal recessive blood disease, enabling to build an accurate, cost-effective and convenient method for prenatal testing for SGDs

Methods

Results

Conclusion