![]() ![]() Universal noninvasive detection of solid organ transplant rejection. Snyder, T.M., Khush, K.K., Valantine, H.A.The HBV drug entecavir - effects on HIV-1 replication and resistance. Maternal viral genotypic zidovudine resistance and infrequent failure of zidovudine therapy to prevent perinatal transmission of human immunodeficiency virus type 1 in pediatric AIDS Clinical Trials Group Protocol 076. Heteroplasmic mitochondrial DNA mutations in normal and tumour cells. Detecting heteroplasmy from high-throughput sequencing of complete human mitochondrial DNA genomes. Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA. Noninvasive prenatal screening for genetic diseases using massively parallel sequencing of maternal plasma DNA. Noninvasive prenatal diagnosis of congenital adrenal hyperplasia using cell-free fetal DNA in maternal plasma. Genomic analysis of fetal nucleic acids in maternal blood. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients. Sputum examination for early detection of lung cancer. High-throughput molecular analysis of urine sediment for the detection of bladder cancer by high-density single-nucleotide polymorphism array. EGFR mutations detected on cytology samples by a centralized laboratory reliably predict response to gefitinib in non-small cell lung carcinoma patients. ![]() BRAF mutation detection in indeterminate thyroid cytology specimens: underlying cytologic, molecular, and pathologic characteristics of papillary thyroid carcinoma. Clinical usefulness of K-ras gene mutation detection and cytology in pancreatic juice in the diagnosis and screening of pancreatic cancer. Utilization of cell-transferred cytologic smears in detection of EGFR and KRAS mutation on adenocarcinoma of lung. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing. High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing. Detecting ultralow-frequency mutations by duplex sequencing. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Ultrasensitive detection of rare mutations using next-generation targeted resequencing. Detection of ultra-rare mutations by next-generation sequencing. Detection and quantification of rare mutations with massively parallel sequencing. Kinde, I., Wu, J., Papadopoulos, N., Kinzler, K.W.Accuracy of next generation sequencing platforms. Fox, E.J., Reid-Bayliss, K.S., Emond, M.J.Keeping up with the next generation: massively parallel sequencing in clinical diagnostics. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) library construction protocol. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients.
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