Mei Baker, MD

Slide of the Week: Mei Baker, MD

Title: Sequencing Library Generation

Legend:  Each probe contains two parts of sequences: one for targeting interested CFTR gene regions, and another for annealing to PCR primers. The DNA templates containing targeted CFTR regions were generated by a sequential process consistent with hybridization, extension, and ligation. The indexes included in PCR primers allow multiplexing of 48 individual DNA samples. The PCR primers are also designed to include P5 and P7, which are necessary for sequencing initiation.

Citations:  Baker MW, Atkins AE, Cordovado SK, Hendrix M, Earley MC, Farrell PM. (2015). Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility studyGenetics in Medicine. 2015 Feb 12. doi: 10.1038/gim.2014.209.

Abstract: Many regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology. Methods: An NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen-positive samples with one CFTR mutation.The NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified. The NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.

About the Lab:  Bakers’s research focuses on public health genetics and genomics, with an emphasis on applying and translating advanced biochemical and molecular technologies into routine newborn screening practice to enable public health laboratories to screen for new conditions and improve screening performance for the existing screened conditions.

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