Long molecule sequencing: a new approach for identification of clinically significant DNA variants in alpha and beta thalassemia carriers.

Abstract Currently, carrier testing for thalassemia requires the application of different molecular tests to provide an accurate genotype. As an alternative methodology, we evaluated long molecule sequencing (LMS) on the PacBio Sequel platform for genotyping carriers of alpha (α) or beta (β) thalassemia. Multiplex long PCR was used to generate representative amplicons for the α (HBA1/2) and β (HBB) gene loci. Following LMS, circular consensus sequencing reads were aligned to the hg19 reference genome and variants called using Free-Bayes software. In a blinded study of 64 known carrier samples, all HBA1/2 and HBB variants detected by LMS were concordant with those independently assigned by targeted PCR assays. For HBA1/2 carrier samples, LMS accurately detected the common --SEA, -α3.7 and -α4.2 deletions and 4 different rare single nucleotide variants (SNVs). For HBB carrier samples, LMS accurately detected the commonest Chinese indel variant c.126_129delCTTT as well as 14 different SNVs/indels and, was able to discriminate compound heterozygous SNVs (trans configuration) and identify variants linked to benign SNPs (cis configuration). Overall, LMS displayed the hallmarks of a scalable, accurate and cost-effective genotyping methodology. With further test coverage to additionally include detection of other clinically-significant HBA1/2 CNVs such as the --THAI, --MED and --FIL deletions, we propose that LMS will eventually serve as a comprehensive method for large-scale thalassemia carrier screening.