Non-invasive prenatal testing of beta-hemoglobinopathies using next generation sequencing, in-silico sequence size selection, and haplotyping.

AIM: To develop a non-invasive prenatal test for beta-hemoglobinopathies based on analyzing maternal plasma by using next generation sequencing. METHODS: We applied next generation sequencing (NGS) of maternal plasma to the non-invasive prenatal testing (NIPT) of autosomal recessive diseases, sickle cell disease and beta-thalassemia. Using the Illumina MiSeq, we sequenced plasma libraries obtained via a Twist Bioscience probe capture panel covering 4 Kb of chromosome 11, including the beta-globin (HBB) gene and >450 genomic single-nucleotide polymorphisms (SNPs) used to estimate the fetal fraction (FF). The FF is estimated by counting paternally transmitted allelic sequence reads present in the plasma but absent in the mother. We inferred fetal beta-globin genotypes by comparing the observed mutation (Mut) and reference (Ref) read ratios to those expected for the three possible fetal genotypes (Mut/Mut; Mut/Ref; Ref/Ref), based on the FF. RESULTS: We bioinformatically enriched the FF by excluding reads over a specified length via in-silico size selection (ISS), favoring the shorter fetal reads, which increased fetal genotype prediction accuracy. Finally, we determined the parental HBB haplotypes, which allowed us to use the read ratios observed at linked SNPs to help predict the fetal genotype at the mutation site(s). We determined HBB haplotypes via Oxford Nanopore MinION sequencing of a 2.2 kb amplicon and aligned these sequences using Soft Genetics' NextGENe LR software. CONCLUSION: The combined use of ISS and HBB haplotypes enabled us to correctly predict fetal genotypes in cases where the prediction based on variant read ratios alone was incorrect.

Noninvasive Prenatal Test for ß-Thalassemia and Sickle Cell Disease Using Probe Capture Enrichment and Next-Generation Sequencing of DNA in Maternal Plasma.

BACKGROUND: Noninvasive prenatal testing (NIPT) of chromosomal aneuploidies based on next-generation sequencing (NGS) analysis of fetal DNA in maternal plasma is well established, but testing for autosomal recessive disorders remains challenging. NGS libraries prepared by probe capture facilitate the analysis of the short DNA fragments plasma. This system has been applied to the ß-hemoglobinopathies to reduce the risk to the fetus. METHOD: Our probe panel captures >4 kb of the HBB region and 435 single-nucleotide polymorphisms (SNPs) used to estimate fetal fraction. Contrived mixtures of DNA samples, plasma, and whole blood samples from 7 pregnant women with ß-thalassemia or sickle cell anemia mutations and samples from the father, sibling, and baby or chorionic villus were analyzed. The fetal genotypes, including point mutations and deletions, were inferred by comparing the observed and expected plasma sequence read ratios, based on fetal fraction, at the mutation site and linked SNPs. Accuracy was increased by removing PCR duplicates and by in silico size selection of plasma sequence reads. A probability was assigned to each of the potential fetal genotypes using a statistical model for the experimental variation, and thresholds were established for assigning clinical status. RESULTS: Using in silico size selection of plasma sequence files, the predicted clinical fetal genotype assignments were correct in 9 of 10 plasma libraries with maternal point mutations, with 1 inconclusive result. For 2 additional plasmas with deletions, the most probable fetal genotype was correct. The ß-globin haplotype determined from linked SNPs, when available, was used to infer the fetal genotype at the mutation site. CONCLUSION: This probe capture NGS assay demonstrates the potential of NIPT for ß-hemoglobinopathies.