Next-generation sequencing (NGS) is present in all fields of life science, which has greatly promoted the development of basic research while being gradually applied in clinical diagnosis. However, the cost and throughput advantages of next-generation sequencing are offset by large tradeoffs with respect to read length and accuracy. Specifically, its high error rate makes it extremely difficult to detect SNPs or low-abundance mutations, limiting its clinical applications, such as pharmacogenomics studies primarily based on SNP and early clinical diagnosis primarily based on low abundance mutations. Currently, Sanger sequencing is still considered to be the gold standard due to its high accuracy, so the results of next-generation sequencing require verification by Sanger sequencing in clinical practice. In order to maintain high quality next-generation sequencing data, a variety of improvements at the levels of template preparation, sequencing strategy and data processing have been developed. This study summarized the general procedures of next-generation sequencing platforms, highlighting the improvements involved in eliminating errors at each step. Furthermore, the challenges and future development of next-generation sequencing in clinical application was discussed.

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.

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BackgroundTraditional farm environments provide protection from allergic diseases. In this study, farm environmental factors were classi ed into three categories: environmental microbes, soil, and organic matter. To explore the impact of soil and environmental microorganisms on gut microbiota and immune function, mice were fed sterilized soil, soil microbes (in lieu of environmental microbes), or non-sterilized soil. ResultsMetagenomic sequencing results showed that the intake of sterile soil while inhaling a small amount of soil microbes in the air, increased gut microbial diversity and the abundance of type III secretion system (T3SS) genes and decreased total serum IgE levels induced by 2-4-dinitro uorobenzene. The intake of soil microbes increased the abundance of genes involved in the metabolism of short-chain fatty acids and amino acid biosynthesis. By contrast, the intake of soil increased gut microbial diversity, the abundance of T3SS genes and related infectious elements, and genes associated with the metabolism of short-chain fatty acids and amino acid biosynthesis and decreased serum IgE levels. The immune function was positively and signi cantly correlated with the bacterial secretion system genes, especially with that of T3SS. ConclusionsAn important mechanism through which farm environments exert a protective effect against allergic diseases could be by serving as a "prebiotic" promoting the reproduction and growth of some intestinal microorganisms that harbor bacterial secretion system genes, especially those of T3SS, whose abundance was positively and signi cantly correlated with innate immune function of mice.

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/μL or 5 CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90-120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens.