RESUMEN
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.
Asunto(s)
Emparejamiento Base , ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Pruebas Prenatales no Invasivas/métodos , Análisis de Secuencia de ADN/métodos , Anemia de Células Falciformes/diagnóstico , Anemia de Células Falciformes/genética , Secuencia de Bases , ADN/química , Humanos , Límite de DetecciónRESUMEN
Perchlorate is a stable anion that has been introduced into the environment through activities related to its production and use as a solid rocket propellant. Perchlorate is thought to transport through soils without being adsorbed; thus, for determination of perchlorate in soil, samples are typically extracted with water prior to analysis. The completeness of extraction depends on perchlorate existing as a free ion within the soil matrix. In this study, perchlorate extraction efficiency was evaluated with five soil types under two different oxygen states. For each soil, 30% (w/w) slurries were prepared and equilibrated under either oxic or anoxic conditions prior to spiking with a stock solution of sodium perchlorate, and the slurries were then maintained for 1-week or 1-month. At the end of the exposure, slurries were centrifuged and separated into aqueous and soil phases. After phase separation, the soil was washed first with deionized water and then with 50mM NaOH, producing second and third aqueous phases, respectively. Perchlorate concentrations in the three aqueous phases were determined using ion chromatography. The results obtained from this study suggest that matrix interference and signal suppression due to high conductivity have greater effects upon observed perchlorate concentrations by ion chromatography than does perchlorate interaction with soil. Thus, a single water extraction is sufficient for quantitative determination of perchlorate in soil.