A novel high-throughput molecular counting method with single base-pair resolution enables accurate single-gene NIPT

Abstract

Next-generation DNA sequencing is currently limited by an inability to accurately count the number of input DNA molecules. Molecular counting is particularly needed when accurate quantification is required for diagnostic purposes, such as in single gene non-invasive prenatal testing (sgNIPT) and liquid biopsy. We developed Quantitative Counting Template (QCT) molecular counting to reconstruct the number of input DNA molecules using sequencing data. We then used QCT molecular counting to develop sgNIPTs of sickle cell disease, cystic fibrosis, spinal muscular atrophy, alpha-thalassemia, and beta-thalassemia. The analytical sensitivity and specificity of sgNIPT was >98% and >99%, respectively. Validation of sgNIPTs was further performed with maternal blood samples collected during pregnancy, and sgNIPTs were 100% concordant with newborn follow-up.

A Method to evaluate analytical sensitivity of qRT-PCR kits for SARS-CoV-2 detection

“…Using a newly developed certi ed reference material, a method to evaluate the sensitivity of qRT-PCR diagnostic kits for SARS-CoV-2 was established. Compared with other reference material derived from invitro transcribed RNA published previously [10][11]27 , the reference material used here was derived from genomic RNA of SARS-CoV-2. Detection sensitivity differed between the three tested kits, stressing the importance of quality control for qRT-PCR assays in the ongoing pandemic.…”

In case you missed it: The Prenatal Diagnosis editors bring you the most significant advances of 2019

“…17 Both of these papers described NIPD in pregnancies at known increased risk; however, 2019 saw the publication of two papers describing the screening of low-risk pregnancies for monogenic disorders. 18,19 These papers have been used to support at least three commercial companies launching platforms publicly available for screening pregnancies at general risk for monogenic disorders.…”

“…18 The second publication also used an NGS-based method for molecular counting to screen for sickle cell disease, cystic fibrosis, spinal muscular atrophy, alpha-thalassemia, and beta-thalassemia. 19 The approach used in this report was to first screen the pregnant mothers' germline DNA, and the cfDNA in maternal plasma is reflex tested in mothers subsequently identified as carriers to determine the fetal genotype. Much of the data reported in this paper comes from modelling experiments using spiked-in genomic DNA, which may be a useful starting point for assay development but, as discussed above, we know that fetal cfDNA is different from maternal cfDNA or postnatal DNA.…”

“…These authors reported excellent performance, but it has the disadvantage of requiring samples from both parents and an affected child or sibling, as it is based on a linkage approach . Both of these papers described NIPD in pregnancies at known increased risk; however, 2019 saw the publication of two papers describing the screening of low‐risk pregnancies for monogenic disorders . These papers have been used to support at least three commercial companies launching platforms publicly available for screening pregnancies at general risk for monogenic disorders.…”

“…The second publication also used an NGS‐based method for molecular counting to screen for sickle cell disease, cystic fibrosis, spinal muscular atrophy, alpha‐thalassemia, and beta‐thalassemia . The approach used in this report was to first screen the pregnant mothers' germline DNA, and the cfDNA in maternal plasma is reflex tested in mothers subsequently identified as carriers to determine the fetal genotype.…”