Evaluation of target-enrichment accompanied by next-generation sequencing for detection of four pathogens in bloodstream infections / 军事医学

Objective To establish an Ampliseq method that combines target-enrichment and the next-generation sequencing for simultaneous detection of Staphylococcus aureus, S.epidermidis, Klebsiella pneumoniae and Listeria monocytogenes in order to provide a fast and accurate means to detect pathogens in bloodstream infections.Methods A method to evaluate the LOD,specificity and sensitivity by constructing simulated samples spiked in known pathogens was established.Results and Conclusion Target-enrichment Ampliseq showed good sensitivity and specificity,and the limit of detection(LOD)was as low as 101CFU/ml.The sensitivity was 95.38%,the specificity was 95.45%and the Kappa value was between 0.839 -1.000.This method can detect S.aureus,S.epidermidis,K.pneumoniae and L.monocytogenes simultaneously in one reaction within 15 hours.

The Role of Next-Generation Sequencing in the Diagnosis of Lysosomal Storage Disorders

Abstract Next-generation sequencing (NGS) panels are used widely in clinical diagnostics to identify genetic causes of various monogenic disease groups including neurometabolic disorders and, more recently, lysosomal storage disorders (LSDs). Many new challenges have been introduced through these new technologies, both at the laboratory level and at the bioinformatics level, with consequences including new requirements for interpretation of results, and for genetic counseling. We review some recent examples of the application of NGS technologies, with purely diagnostic and with both diagnostic and research aims, for establishing a rapid genetic diagnosis in LSDs. Given that NGS can be applied in a way that takes into account the many issues raised by international consensus guidelines, it can have a significant role even early in the course of the diagnostic process, in combination with biochemical and clinical data. Besides decreasing the delay in diagnosis for many patients, a precise molecular diagnosis is extremely important as new therapies are becoming available within the LSD spectrum for patients who share specific types of mutations. A genetic diagnosis is also the prerequisite for genetic counseling, family planning, and the individual choice of reproductive options in affected families.

New Lung Cancer Panel for High-Throughput Targeted Resequencing

We present a new next-generation sequencing-based method to identify somatic mutations of lung cancer. It is a comprehensive mutation profiling protocol to detect somatic mutations in 30 genes found frequently in lung adenocarcinoma. The total length of the target regions is 107 kb, and a capture assay was designed to cover 99% of it. This method exhibited about 97% mean coverage at 30x sequencing depth and 42% average specificity when sequencing of more than 3.25 Gb was carried out for the normal sample. We discovered 513 variations from targeted exome sequencing of lung cancer cells, which is 3.9-fold higher than in the normal sample. The variations in cancer cells included previously reported somatic mutations in the COSMIC database, such as variations in TP53, KRAS, and STK11 of sample H-23 and in EGFR of sample H-1650, especially with more than 1,000x coverage. Among the somatic mutations, up to 91% of single nucleotide polymorphisms from the two cancer samples were validated by DNA microarray-based genotyping. Our results demonstrated the feasibility of high-throughput mutation profiling with lung adenocarcinoma samples, and the profiling method can be used as a robust and effective protocol for somatic variant screening.