OBJECTIVETo establish a hyperphenylalaninemia related genes screening method using Ion Torrent Personal Genome Machine (PGM) for early detection and differential diagnosis of hyperphenylalaninemia (HPA).

METHODSThree children with known HPA mutations and a healthy control were used for setting up the method. Ten children with HPA with known mutations were recruited for validating the method. Ion Ampliseq PCR was used to amplify the 5' and 3' untranslated region, coding sequence, and flanking introns of PAH, GCH1, PTS, QDPR, and PCBD1 genes. After the enrichment with the Ion OneTouch system, the products were sequenced by PGM. Data from the PGM were processed with Torrent Suite v2.2 software package. All variations were confirmed by Sanger sequencing.

RESULTSFor the 4 samples, the PGM output was 94.22 Mb, with approximately 99.5% of reads mapping to the target regions. Among these samples, we detected 74 variations (28 positions) including 6 known mutations. Compared with database and results of Sanger sequencing, 55 (18 positions) polymorphisms and 13 (4 positions) false positive calls were confirmed. For the 10 samples, all the known mutations were successfully identified.

CONCLUSIONIon Torrent PGM sequencing is suitable for screening genetic mutation underlying HPA from the perspective of metabolic pathways, which can meet the clinical demand for individualized diagnosis and treatment.

No abstract available.
DNA, Neoplasm/genetics ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Sequence Analysis, DNA/methods ; Urologic Neoplasms/*genetics

Infectious diseases are commonly seen in clinical practice, and pathogen diagnosis is the key link in diagnosis and treatment; however, conventional pathogen detection methods cannot meet clinical needs due to time-consuming operation and low positive rate. As a new pathogen detection method, metagenomic next-generation sequencing (mNGS) has a wide detection range and can detect bacteria, viruses, fungi, parasites, rare pathogens, and even unknown pathogens. The technique of mNGS is unbiased and can rapidly, efficiently, and accurately obtain all nucleic acid information in test samples, analyze pathogens, and guide clinical diagnosis and treatment, thereby playing an important role in complicated infectious diseases. This article reviews the diagnostic advantages and clinical value of mNGS in bacterial, fungal, viral, and parasitic infections.
Bacteria ; Communicable Diseases/diagnosis* ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Metagenomics/methods* ; Sensitivity and Specificity

OBJECTIVES: To study the application value of metagenomic next-generation sequencing (mNGS) in children with severe infectious diseases. METHODS: An analysis was performed on the clinical data and laboratory test results of 29 children with severe infection who were admitted to the Second Affiliated Hospital of Wenzhou Medical University from June 2018 to December 2020. Conventional pathogen culture was performed for the 29 specimens (27 peripheral blood specimens and 2 pleural effusion specimens) from the 29 children, and mNGS pathogen detection was performed at the same time. RESULTS: Among the 29 children, 2 tested positive by conventional pathogen culture with 2 strains of pathogen, and the detection rate was 7% (2/29); however, 20 children tested positive by mNGS with 38 strains of pathogen, and the detection rate was 69% (20/29). The pathogen detection rate of mNGS was significantly higher than that of conventional pathogen culture (P<0.05), and mNGS could detect the viruses, fungi, and other special pathogens that conventional pathogen culture failed to detect, such as Orientia tsutsugamushi. The univariate analysis showed that gender, routine blood test results, C-reactive protein, procalcitonin, D-dimer, radiological findings, and whether antibiotics were used before admission did not affect the results of mNGS (P>0.05). CONCLUSIONS Compared with conventional pathogen culture, mNGS is more sensitive for pathogen detection, with fewer interference factors. Therefore, it is a better pathogenic diagnosis method for severe infectious diseases in children.
Anti-Bacterial Agents ; Child ; Communicable Diseases ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Metagenomics/methods* ; Sensitivity and Specificity

"Active health" has been emphasized in "Healthy China 2030" in dealing with the challenges of population aging, so the anti-aging strategies are requires to be more precise and effective at both individual and population levels. Aging is influenced by both genetic and environmental factors. In the recent 20 years, the research of genetics of human ageing has been greatly facilitated owning to the development of high-throughput sequencing techniques, statistical methodology for multi-omics data, as well as the growing qualified evidence of large-scale population-based genomic research. This paper provides a review of genome-wide association research of aging.
Aging/genetics* ; Genome-Wide Association Study/methods* ; Genomics/methods* ; High-Throughput Nucleotide Sequencing ; Humans ; Phenotype

The Sanger sequencing techniques, also known as the first-generation sequencing techniques and the gold standard of sequencing, have promoted the completion of "working draft" of the human genome, but the disadvantages of low throughput and high cost limit its large-scale application. The second-generation sequencing techniques, also known as the next-generation sequencing techniques, have widely used in basic research and clinical application because of its high throughput and low cost, but the short reads has always been an unavoidable shortcoming. Then, the emergence of the third-generation sequencing techniques, with the long reads, provides new technology selection for the analysis of complex repetitive regions on genome sequences and the assembly of high-quality genomes. In recent years, the third-generation sequencing techniques have been further developed, and have gradually demonstrated the clinical application value. This article reviewed the research progress and clinical application of the third-generation sequencing techniques.
Genome ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Sequence Analysis, DNA/methods*

The quality of traditional Chinese medicines (TCMs) has been mainly evaluated based on chemical ingredients, yet recently more attentions have been paid on biological ingredients, especially for pill-based preparations. It is a key approach to establish a fast, accurate and systematic method of biological ingredient analysis for realization of modernization, industrialization and internationalization of TCMs. The biological ingredient analysis of TCM preparations could be abstracted as the identification of multiple species from a biological mixture. The metagenomic approach based on high-throughput-sequencing (HTS) and big-data-mining has been considered as one of the most effective methods for multiple species analysis of a biological mixture, which would also be helpful for the analysis of biological ingredients in TCMs. Simultaneous identification of diverse species, including the prescribed species, adulterants, toxic species, protected species and even the biological impurities introduced through production process, could be achieved by selecting appropriate DNA biomarkers, as well as applying large-scale sequence comparison and data mining. By this approach, it is prospective to offer an evaluation basis for the effectiveness, safety and legality of TCM preparations.
Biological Products ; chemistry ; Data Mining ; High-Throughput Nucleotide Sequencing ; Medicine, Chinese Traditional ; Metagenomics ; methods

PURPOSE: We evaluated the clinical role of rapid next-generation sequencing (NGS) for identifying BRCA1/2 mutations compared to traditional Sanger sequencing. METHODS: Twenty-four paired samples from 12 patients were analyzed in this prospective study to compare the performance of NGS to the Sanger method. Both NGS and Sanger sequencing were performed in 2 different laboratories using blood samples from patients with breast cancer. We then analyzed the accuracy of NGS in terms of variant calling and determining concordance rates of BRCA1/2 mutation detection. RESULTS: The overall concordance rate of BRCA1/2 mutation identification was 100%. Variants of unknown significance (VUS) were reported in two cases of BRCA1 and 3 cases of BRCA2 after Sanger sequencing, whereas NGS reported only 1 case of BRCA1 VUS, likely due to differences in reference databases used for mutation identification. The median turnaround time of Sanger sequencing was 22 days (range, 14–26 days), while the median time of NGS was only 6 days (range, 3–21 days). CONCLUSION: NGS yielded comparably accurate results to Sanger sequencing and in a much shorter time with respect to BRCA1/2 mutation identification. The shorter turnaround time and higher accuracy of NGS may help clinicians make more timely and informed decisions regarding surgery or neoadjuvant chemotherapy in patients with breast cancer.
Breast Neoplasms* ; Breast* ; Clinical Decision-Making* ; Drug Therapy ; High-Throughput Nucleotide Sequencing ; Humans ; Methods ; Prospective Studies

OBJECTIVETo detect the pathogenic mutation in a patient with methylmalonic acidemia using IonTorrent Personal Genome Machine (PGM) and assess the feasibility of such technology for analyzing complex monogenic diseases.

METHODSPeripheral blood sample was collected from the patient. Genomic DNA was isolated using a standard method and subjected to targeted sequencing using an Ion Ampliseq Inherited Disease Panel. DNA fragment was ligated with a barcoded sequencing adaptor. Template preparation, emulsion PCR, and Ion Sphere Particles enrichment were carried out using the Ion One Touch system. Data from the PGM runs were processed using Ion Torrent Suite 3.2 software to generate sequence reads. All variants were filtered against dbSNPl37. DNA sequences were visualized with an Integrated Genomics Viewer.

RESULTSAfter data analysis and database filtering, a previously reported nonsense mutation, c.586C>T (p.R196X), and a novel mutation c.898C>T (p.R300X) were identified in the MMAA gene in this patient. Both mutations were verified by conventional Sanger sequencing.

CONCLUSIONPathogenic MMAA mutations have been identified in a patient with methylmalonic acidemia. This new-generation targeted sequencing on the PGM sequencers can be applied for genetic diagnosis of hereditary diseases.

With the development and improvement of new sequencing technology, next-generation sequencing (NGS) has been applied increasingly in cancer genomics research over the past decade. More recently, NGS has been adopted in clinical oncology to advance personalized treatment of cancer. NGS is used to identify novel and rare cancer mutations, detect familial cancer mutation carriers, and provide molecular rationale for appropriate targeted therapy. Compared to traditional sequencing, NGS holds many advantages, such as the ability to fully sequence all types of mutations for a large number of genes (hundreds to thousands) in a single test at a relatively low cost. However, significant challenges, particularly with respect to the requirement for simpler assays, more flexible throughput, shorter turnaround time, and most importantly, easier data analysis and interpretation, will have to be overcome to translate NGS to the bedside of cancer patients. Overall, continuous dedication to apply NGS in clinical oncology practice will enable us to be one step closer to personalized medicine.
High-Throughput Nucleotide Sequencing ; economics ; methods ; Humans ; Mutation ; Neoplasms ; genetics ; Precision Medicine ; Sequence Analysis, DNA