scholarly journals High-throughput next-generation sequencing for identifying pathogens during early-stage post-lung transplantation

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Qiao-yan Lian ◽  
Ao Chen ◽  
Jian-heng Zhang ◽  
Wei-jie Guan ◽  
Xin Xu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Lung Transplantation ◽  
High Throughput ◽  
Pathogen Detection ◽  
Pulmonary Infection ◽  
Early Stage ◽  
Next Generation ◽  
Treatment Regimens ◽  
Microbiological Testing ◽  
Generation Sequencing

Abstract Background High-throughput next-generation sequencing (HT-NGS) has the potential to detect a large variety of pathogens; however, the application of HT-NGS in lung transplant (LTx) recipients remains limited. We aimed to evaluate the value of HT-NGS for pathogen detection and diagnosis of pulmonary infection during early-stage post-lung transplantation. Methods In this retrospective study, we enrolled 51 LTx recipients who underwent lung transplantation between January 2020 and December 2020. Bronchoalveolar lavage fluid (BALF) samples were collected for the detection of pathogens using both HT-NGS and conventional microbiological testing. The detection of pathogens and diagnostic performance of HT-NGS were compared with that of conventional methods. Results HT-NGS provided a higher positive rate of pathogen detection than conventional microbiological testing (88.24% vs. 76.47%). The most common bacteria detected via HT-NGS during early-stage post-lung transplantation were Enterococcus, Staphylococcus, Pseudomonas and Klebsiella, while all fungi were Candida and all viruses were Herpesvirus. Uncommon pathogens, including Strongyloides, Legionella, and Mycobacterium abscesses were identified by HT-NGS. The sensitivity of HT-NGS for diagnosing pulmonary infection was significantly higher than that of conventional microbiological testing (97.14% vs. 68.57%; P < 0.001). For three LTx recipients, treatment regimens were adjusted according to the results of HT-NGS, leading to a complete recovery. Conclusion HT-NGS is a highly sensitive technique for pathogen detection, which may provide diagnostic advantages, especially in LTx recipients, contributing to the optimization of treatment regimens against pulmonary infection during early-stage post-lung transplantation.

miRNA profiling in resectable NSCLC by multiplex next-generation sequencing.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7060-7060
Author(s):  
Sandra Gallach ◽  
Eloisa Jantus-Lewintre ◽  
David Montaner ◽  
Marta Usó ◽  
Elena Sanmartin ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Mirna Expression ◽  
Early Stage ◽  
Normal Lung ◽  
Next Generation ◽  
Early Stage Nsclc ◽  
Nsclc Patients ◽  
Generation Sequencing ◽  
Independent Cohort

7060 Background: Early-stage NSCLC has a relapse rate around 40% within 5 years. With the advent of microRNA (miRNA), which seems to regulate many genes critical for tumorigenesis, there is a growing interest in the characterization of miRNAome in NSCLC specimens and to correlate them with prognosis. Next generation sequencing is a useful tool to study the miRNA content of solid tumors. Here, we applied high-throughput SOLiD transcriptome sequencing to study miRNAs expression in a cohort of early-stage NSCLC patients (tumor vs normal lung). Methods: RNA was isolated from frozen lung specimens (tumor and normal lung) from resectable NSCLC patients (n=35). Samples with a RIN ≥ 7 were analyzed and enriched in the miRNA fraction. miRNAs were sequencing using a bar-code multiplex SOLiD protocol. Data normalization was carried out by rescaling all data according to their counts. Readings were mapped against mature and no-mature miRNAs using miRBase. Statistical analysis was performed with CLCbio software and considered significant when p-adj<0.005. Results: Using the SOLiD high throughput sequencing, we performed a systemic miRNA expression profiling analysis of paired samples (tumor vs normal lung). A total of 1268 miRNAs (mature and no-mature) have been detected in at least one sample. The differential expression between normal and tumor samples shown that 6 miRNAs (miR-193b, miR-182, miR-96, miR-148a, miR-299, miR-590) were upregulated and 7 (miR-145, miR-133a, miR-218, miR-125a, miR-30a, miR-126 and miR-139) were down-regulated significantly in tumor samples compared with normal lung tissues. We are performing studies using qRT-PCR in an independent cohort to further validate these findings. Conclusions: The deep sequencing technology used for differential miRNA expression is useful and novel. The use of barcoding allows multiplexing and lowers cost per sample. Several miRNAs were differentially expressed between tumor and normal tissue, but this point needs to be further validated in an independent cohort. Supported by grants TRA09-0132 (MICINN) and RD06/0020/1024 (ISCIII).

scholarly journals Improving Pulmonary Infection Diagnosis with Metagenomic Next Generation Sequencing

2021 ◽  
Vol 10 ◽  
Author(s):  
Yi-Yi Qian ◽  
Hong-Yu Wang ◽  
Yang Zhou ◽  
Hao-Cheng Zhang ◽  
Yi-Min Zhu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pathogen Detection ◽  
Pulmonary Infection ◽  
Diagnostic Methods ◽  
Diagnostic Efficiency ◽  
Pulmonary Infections ◽  
Next Generation ◽  
Standard Polymerase Chain Reaction ◽  
Conventional Methods ◽  
Generation Sequencing

Pulmonary infections are among the most common and important infectious diseases due to their high morbidity and mortality, especially in older and immunocompromised individuals. However, due to the limitations in sensitivity and the long turn-around time (TAT) of conventional diagnostic methods, pathogen detection and identification methods for pulmonary infection with greater diagnostic efficiency are urgently needed. In recent years, unbiased metagenomic next generation sequencing (mNGS) has been widely used to detect different types of infectious pathogens, and is especially useful for the detection of rare and newly emergent pathogens, showing better diagnostic performance than traditional methods. There has been limited research exploring the application of mNGS for the diagnosis of pulmonary infections. In this study we evaluated the diagnostic efficiency and clinical impact of mNGS on pulmonary infections. A total of 100 respiratory samples were collected from patients diagnosed with pulmonary infection in Shanghai, China. Conventional methods, including culture and standard polymerase chain reaction (PCR) panel analysis for respiratory tract viruses, and mNGS were used for the pathogen detection in respiratory samples. The difference in the diagnostic yield between conventional methods and mNGS demonstrated that mNGS had higher sensitivity than traditional culture for the detection of pathogenic bacteria and fungi (95% vs 54%; p&lt;0.001). Although mNGS had lower sensitivity than PCR for diagnosing viral infections, it identified 14 viral species that were not detected using conventional methods, including multiple subtypes of human herpesvirus. mNGS detected viruses with a genome coverage &gt;95% and a sequencing depth &gt;100× and provided reliable phylogenetic and epidemiological information. mNGS offered extra benefits, including a shorter TAT. As a complementary approach to conventional methods, mNGS could help improving the identification of respiratory infection agents. We recommend the timely use of mNGS when infection of mixed or rare pathogens is suspected, especially in immunocompromised individuals and or individuals with severe conditions that require urgent treatment.

scholarly journals The Application of Metagenomic Next-Generation Sequencing in Detection of Pathogen in Bronchoalveolar Lavage Fluid and Sputum Samples of Patients with Pulmonary Infection

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Wanghui Shi ◽  
Shanshan Zhu
Keyword(s):  
Next Generation Sequencing ◽  
Bronchoalveolar Lavage ◽  
Pathogen Detection ◽  
Bronchoalveolar Lavage Fluid ◽  
Pulmonary Infection ◽  
Lavage Fluid ◽  
Microbial Culture ◽  
Next Generation ◽  
Two Samples ◽  
Generation Sequencing

Objective. To uncover the application value of metagenomic next-generation sequencing (mNGS) in the detection of pathogen in bronchoalveolar lavage fluid (BALF) and sputum samples. Methods. Totally, 32 patients with pulmonary infection were included. Pathogens in BALF and sputum samples were tested simultaneously by routine microbial culture and mNGS. Main infected pathogens (bacteria, fungi, and viruses) and their distribution in BALF and sputum samples were analyzed. Moreover, the diagnostic performance of mNGS in paired BALF and sputum samples was assessed. Results. The pathogen culture results were positive in 9 patients and negative in 13 patients. No statistical differences were recorded on the sensitivity (78.94% vs. 63.15%, p = 0.283 ) and specificity (62.50% vs. 75.00%, p = 0.375 ) of mNGS diagnosis in bacteria and fungus in two types of samples. As shown in mNGS detection, 10 patients’ two samples were both positive, 13 patients’ two samples were both negative, 7 patients were only positive in BALF samples, and 2 patients’ sputum samples were positive. Main viruses mNGS detected were EB virus, human adenovirus 5, herpes simplex virus type 1, and human cytomegalovirus. Kappa consensus analysis indicated that mNGS showed significant consistency in detecting pathogens in two samples, no matter bacteria ( p < 0.001 ), fungi ( p = 0.026 ), or viruses ( p = 0.008 ). Conclusion. mNGS showed no statistical differences in sensitivity and specificity of pathogen detection in BALF and sputum samples. Under certain conditions, sputum samples might be more suitable for pathogen detection because of invasiveness of BALF samples.

Next-Generation Sequencing: An Emerging Tool for Drug Designing

2019 ◽  
Vol 25 (31) ◽  
pp. 3350-3357 ◽  
Author(s):  
Pooja Tripathi ◽  
Jyotsna Singh ◽  
Jonathan A. Lal ◽  
Vijay Tripathi
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Large Scale ◽  
Massively Parallel Sequencing ◽  
Genomic Research ◽  
Biological Research ◽  
Next Generation ◽  
Human Welfare ◽  
Drug Designing ◽  
Generation Sequencing

Background: With the outbreak of high throughput next-generation sequencing (NGS), the biological research of drug discovery has been directed towards the oncology and infectious disease therapeutic areas, with extensive use in biopharmaceutical development and vaccine production. Method: In this review, an effort was made to address the basic background of NGS technologies, potential applications of NGS in drug designing. Our purpose is also to provide a brief introduction of various Nextgeneration sequencing techniques. Discussions: The high-throughput methods execute Large-scale Unbiased Sequencing (LUS) which comprises of Massively Parallel Sequencing (MPS) or NGS technologies. The Next geneinvolved necessarily executes Largescale Unbiased Sequencing (LUS) which comprises of MPS or NGS technologies. These are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. Conclusion: Analysis of NGS data unravels important clues in the quest for the treatment of various lifethreatening diseases and other related scientific problems related to human welfare.

scholarly journals Panel of serum miRNAs as potential non-invasive biomarkers for pancreatic ductal adenocarcinoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Imteyaz Ahmad Khan ◽  
Safoora Rashid ◽  
Nidhi Singh ◽  
Sumaira Rashid ◽  
Vishwajeet Singh ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Early Stage ◽  
Ductal Adenocarcinoma ◽  
Next Generation ◽  
Serum Samples ◽  
Tissue Samples ◽  
Non Invasive ◽  
Specific Symptoms ◽  
Generation Sequencing

AbstractEarly-stage diagnosis of pancreatic ductal adenocarcinoma (PDAC) is difficult due to non-specific symptoms. Circulating miRNAs in body fluids have been emerging as potential non-invasive biomarkers for diagnosis of many cancers. Thus, this study aimed to assess a panel of miRNAs for their ability to differentiate PDAC from chronic pancreatitis (CP), a benign inflammatory condition of the pancreas. Next-generation sequencing was performed to identify miRNAs present in 60 FFPE tissue samples (27 PDAC, 23 CP and 10 normal pancreatic tissues). Four up-regulated miRNAs (miR-215-5p, miR-122-5p, miR-192-5p, and miR-181a-2-3p) and four down-regulated miRNAs (miR-30b-5p, miR-216b-5p, miR-320b, and miR-214-5p) in PDAC compared to CP were selected based on next-generation sequencing results. The levels of these 8 differentially expressed miRNAs were measured by qRT-PCR in 125 serum samples (50 PDAC, 50 CP, and 25 healthy controls (HC)). The results showed significant upregulation of miR-215-5p, miR-122-5p, and miR-192-5p in PDAC serum samples. In contrast, levels of miR-30b-5p and miR-320b were significantly lower in PDAC as compared to CP and HC. ROC analysis showed that these 5 miRNAs can distinguish PDAC from both CP and HC. Hence, this panel can serve as a non-invasive biomarker for the early detection of PDAC.

scholarly journals Advances in genetics and molecular breeding of three legume crops of semi-arid tropics using next-generation sequencing and high-throughput genotyping technologies

2012 ◽  
Vol 37 (5) ◽  
pp. 811-820 ◽  
Author(s):  
Rajeev K Varshney ◽  
Himabindu Kudapa ◽  
Manish Roorkiwal ◽  
Mahendar Thudi ◽  
Manish K Pandey ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Molecular Breeding ◽  
Next Generation ◽  
Legume Crops ◽  
Generation Sequencing ◽  
Semi Arid

scholarly journals A Comparison of Blood Pathogen Detection Among Droplet Digital PCR, Metagenomic Next-Generation Sequencing, and Blood Culture in Critically Ill Patients With Suspected Bloodstream Infections

2021 ◽  
Vol 12 ◽  
Author(s):  
Bangchuan Hu ◽  
Yue Tao ◽  
Ziqiang Shao ◽  
Yang Zheng ◽  
Run Zhang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Blood Culture ◽  
Critically Ill ◽  
Critically Ill Patients ◽  
Pathogen Detection ◽  
Bloodstream Infections ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Next Generation ◽  
Generation Sequencing

Metagenomic next-generation sequencing (mNGS) and droplet digital PCR (ddPCR) have recently demonstrated a great potential for pathogen detection. However, few studies have been undertaken to compare these two nucleic acid detection methods for identifying pathogens in patients with bloodstream infections (BSIs). This prospective study was thus conducted to compare these two methods for diagnostic applications in a clinical setting for critically ill patients with suspected BSIs. Upon suspicion of BSIs, whole blood samples were simultaneously drawn for ddPCR covering 20 common isolated pathogens and four antimicrobial resistance (AMR) genes, mNGS, and blood culture. Then, a head-to-head comparison was performed between ddPCR and mNGS. A total of 60 episodes of suspected BSIs were investigated in 45 critically ill patients, and ddPCR was positive in 50 (83.3%), mNGS in 41 (68.3%, not including viruses), and blood culture in 10 (16.7%) episodes. Of the 10 positive blood cultures, nine were concordantly identified by both mNGS and ddPCR methods. The head-to-head comparison showed that ddPCR was more rapid (~4 h vs. ~2 days) and sensitive (88 vs. 53 detectable pathogens) than mNGS within the detection range of ddPCR, while mNGS detected a broader range of pathogens (126 vs. 88 detectable pathogens, including viruses) than ddPCR. In addition, a total of 17 AMR genes, including 14 blaKPC and 3 mecA genes, were exclusively identified by ddPCR. Based on their respective limitations and strengths, the ddPCR method is more useful for rapid detection of common isolated pathogens as well as AMR genes in critically ill patients with suspected BSI, whereas mNGS testing is more appropriate for the diagnosis of BSI where classic microbiological or molecular diagnostic approaches fail to identify causative pathogens.

scholarly journals Molecular Identification and Genetic Characterization of Early-Stage Multiple Primary Lung Cancer by Large-Panel Next-Generation Sequencing Analysis

2021 ◽  
Vol 11 ◽  
Author(s):  
Guotian Pei ◽  
Mingwei Li ◽  
Xianjun Min ◽  
Qiang Liu ◽  
Dasheng Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Early Stage ◽  
Primary Lung Cancer ◽  
Advanced Stage ◽  
Next Generation ◽  
Molecular Method ◽  
Multiple Primary ◽  
Large Panel ◽  
Generation Sequencing

ObjectiveThe incidence of early stage multiple primary lung cancer (MPLC) has been increasing in recent years, while the ideal strategy for its diagnosis and treatment remains controversial. The present study conducted genomic analysis to identify a new molecular classification method for accurately predicting the diagnosis and therapy for patients with early stage MPLC.MethodsA total of 240 tissue samples from 203 patients with multiple-non-small-cell lung cancers (NSCLCs) (n = 30), early stage single-NSCLC (Group A, n = 94), and advanced-stage NSCLC (Group B, n = 79) were subjected to targeted multigene panel sequencing.ResultsThirty patients for whom next-generation sequencing was performed on &gt;1 tumor were identified, yielding 45 tumor pairs. The frequencies of EGFR, TP53, RBM10, ERBB2, and CDKN2A mutations exhibited significant differences between early and advanced-stage NSCLCs. The prevalence of the EGFR L858R mutation in early stage NSCLC was remarkably higher than that in advanced-stage NSCLC (P = 0.047). The molecular method classified tumor pairs into 26 definite MPLC tumors and four intrapulmonary metastasis (IM) tumors. A high rate of discordance in driver genetic alterations was found in the different tumor lesions of MPLC patients. The prospective Martini histologic prediction of MPLC was discordant with the molecular method for three patients (16.7%), particularly in the prediction of IM (91.7% discordant).ConclusionsComprehensive molecular evaluation allows the unambiguous delineation of clonal relationships among tumors. In comparison, the Martini and Melamed criteria have notable limitations in the recognition of IM. Our results support the adoption of a large panel to supplement histology for strongly discriminating NSCLC clonal relationships in clinical practice.

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