digitalMLPA EZtec-MS: A new highly multiplexed DNA probe-based technique for neonatal screening

Decreasing DNA sequencing costs has paved the way to the use of next generation sequencing (NGS) to expand the number of conditions tested in routine newborn screening (NBS) programs. Expanding NBS programs by NGS, however, raises technical, ethical and economic concerns that are important for low-middle income countries or countries that are hesitant to use sequencing in a public health setting. qPCR-based DNA analyses have already been implemented in routine NBS programs; however, qPCR cannot be highly multiplexed. digitalMLPA (dMLPA) is a highly multiplexed probe-based DNA technique. Here, we describe the new dMLPA EZtec-MS technique that was specifically developed for use on dried blot spots (DBS) and can target up to 1200 DNA sequences representing different variant types, allowing high-throughput testing of crude DNA extracts prepared from a single DBS punch. The assay was validated using positive DNA samples and crude DNA extracts from DBS of 2069 negatively screened Philippine newborns. The EZtec-MS assay used in this study successfully detected copy number variants, (including complex genomic regions (SMN1/2, HBA1/2)), inversions (F8), methylation (imprinting diseases, Fragile X), single nucleotide variants (including ATP7B, CTNS, MT-RNR1), and low copy number sequences (TRECs for SCID, cytomegalovirus) in a single reaction in both positive DNA and crude DNA extracts from DBS. The results also demonstrated that dMLPA EZtec-MS is a high-throughput method, suitable for DBS, and has a relatively short turn-around time of 36-40 h. Our findings indicate that the dMLPA EZtec-MS technique is a promising versatile tool for DNA-based NBS programs.