scholarly journals First-Catch, Mid-Stream and Catheterised urine: A Comparative Study of Male Urinary Microbiome by Expanded Quantitative Urine Culture and Next-Generation Sequencing

2021 ◽  
Author(s):  
Jan Hrbacek ◽  
Daniel Morais ◽  
Pavel Cermak ◽  
Vitezslav Hanacek ◽  
Roman Zachoval
Keyword(s):  
Next Generation Sequencing ◽  
Urinary Bladder ◽  
Urine Sample ◽  
Urine Culture ◽  
Urine Samples ◽  
Next Generation ◽  
Sample Collection ◽  
Microbial Composition ◽  
Urinary Microbiota ◽  
Generation Sequencing

Abstract BackgroundNumerous studies have emerged in the past decade investigating human urinary microbiota. Alterations in the microbial composition of urine have been linked to structural and functional abnormalities of the lower urinary tract. There has been considerable variation in the methodology of the studies published so far including the cornerstone of any biomedical analysis: sample collection. The aim of this study was to describe the urinary microbiota of first-catch voided urine (FCU), mid-stream voided urine (MSU) and aseptically catheterised urine in men and find the most suitable approach to urine sample collection for the purpose of male urinary microbiota investigations.ResultsForty-nine men (mean age 71.3 years) undergoing endoscopic procedures in our Department of Urology were enrolled in the study. Each of them contributed three samples: first-catch urine (FCU), mid-stream urine (MSU) and a catheterised urine sample. The samples were subjected to next-generation sequencing (NGS, n=35) and expanded quantitative urine culture (EQUC, n=31). Using NGS, Bacteroidetes, Firmicutes, and Proteobacteria were the most abundant phyla in our population. The most abundant genera (in order of relative abundance) included: Prevotella, Veillonella, Streptococcus, Porphyromonas, Campylobacter, Pseudomonas, Staphylococcus, Ezakiella, Escherichia and Dialister. Eighty-two of 105 samples were dominated by a single genus. FCU, MSU and catheterised urine samples differed significantly using ANOVA in three out of five alpha-diversity measures (p<0.05): estimated number of operational taxonomic units, Chao1 and abundance-based coverage estimators. There were no differences found in Simpson and Shannon indices. Beta-diversity comparisons using the PIME method (Prevalence Interval for Microbiome Evaluation) resulted in clear clustering of urine samples according to the mode of sampling.EQUC detected cultivable bacteria in 30/31 (97%) FCU and 27/31 (87%) MSU samples. Only 4/31 (13%) of catheterised urine samples showed bacterial growth.ConclusionsUrine samples obtained by transurethral catheterisation under aseptic conditions differ from spontaneously voided urine samples and represent a better reflection of urinary bladder microbiota. Catheterised urine is the most appropriate way to sample urine in future studies of urinary bladder pathological conditions and their relation to the urinary microbiota.

scholarly journals Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

2017 ◽  
Author(s):  
Taha Soliman ◽  
Sung-Yin Yang ◽  
Tomoko Yamazaki ◽  
Holger Jenke-Kodama
Keyword(s):  
Next Generation Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Soil Microbial Communities ◽  
Next Generation ◽  
Microbial Composition ◽  
Okinawa Island ◽  
The Impact ◽  
Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P <0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

scholarly journals Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

2017 ◽  
Author(s):  
Taha Soliman ◽  
Sung-Yin Yang ◽  
Tomoko Yamazaki ◽  
Holger Jenke-Kodama
Keyword(s):  
Next Generation Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Soil Microbial Communities ◽  
Next Generation ◽  
Microbial Composition ◽  
Okinawa Island ◽  
The Impact ◽  
Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P <0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

scholarly journals Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4178 ◽  
Author(s):  
Taha Soliman ◽  
Sung-Yin Yang ◽  
Tomoko Yamazaki ◽  
Holger Jenke-Kodama
Keyword(s):  
Next Generation Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Soil Microbial Communities ◽  
Next Generation ◽  
Microbial Composition ◽  
Okinawa Island ◽  
The Impact ◽  
Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil®DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin®Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA;P < 0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

scholarly journals Detection of Pathogenic Microbe Composition Using Next-Generation Sequencing Data

2020 ◽  
Vol 11 ◽  
Author(s):  
Haiyong Zhao ◽  
Shuang Wang ◽  
Xiguo Yuan
Keyword(s):  
Next Generation Sequencing ◽  
Computational Method ◽  
Superior Performance ◽  
Next Generation Sequencing Data ◽  
Support Vector ◽  
Next Generation ◽  
Sequencing Data ◽  
Microbial Composition ◽  
High Resolution Data ◽  
Generation Sequencing

Next-generation sequencing (NGS) technologies have provided great opportunities to analyze pathogenic microbes with high-resolution data. The main goal is to accurately detect microbial composition and abundances in a sample. However, high similarity among sequences from different species and the existence of sequencing errors pose various challenges. Numerous methods have been developed for quantifying microbial composition and abundance, but they are not versatile enough for the analysis of samples with mixtures of noise. In this paper, we propose a new computational method, PGMicroD, for the detection of pathogenic microbial composition in a sample using NGS data. The method first filters the potentially mistakenly mapped reads and extracts multiple species-related features from the sequencing reads of 16S rRNA. Then it trains an Support Vector Machine classifier to predict the microbial composition. Finally, it groups all multiple-mapped sequencing reads into the references of the predicted species to estimate the abundance for each kind of species. The performance of PGMicroD is evaluated based on both simulation and real sequencing data and is compared with several existing methods. The results demonstrate that our proposed method achieves superior performance. The software package of PGMicroD is available at https://github.com/BDanalysis/PGMicroD.

Diagnosis of Taenia solium infections based on “mail order” RNA-sequencing of single tapeworm egg isolates from stool samples v1

2021 ◽  
Author(s):  
Henrik Sadlowski ◽  
Veronika Schmidt ◽  
Jonathan Hiss ◽  
Christian G. Schneider ◽  
Gideon Zulu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Taenia Solium ◽  
Mail Order ◽  
Next Generation ◽  
Sample Collection ◽  
Additional Information ◽  
Stool Samples ◽  
Sequencing Facility ◽  
Generation Sequencing

Here we present a detailed protocol for the identification of Taenia solium based on the few Taenia spp. eggs found in diagnostic stool samples. Our approach is based on "mail order" RNA sequencing of single eggs and can be performed in laboratories equipped with basic tools such as a microscope, a Bunsen burner, and access to an international post office for shipping samples to a next-generation sequencing facility. This protocol describes sample collection and transport, isolation of individual Taenia spp. eggs, reliable disruption of individual Taenia eggs, and important considerations for shipping samples to a next-generation sequencing facility. We provide images and videos to help prepare the tools needed for the protocol. Additional information on our rationale for designing the critical steps can help implement the protocol in new environments.

Next-generation sequencing not superior to culture in periprosthetic joint infection diagnosis

2021 ◽  
Vol 103-B (1) ◽  
pp. 26-31
Author(s):  
Beau J. Kildow ◽  
Sean P. Ryan ◽  
Richard Danilkowicz ◽  
Alexander L. Lazarides ◽  
Colin Penrose ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Periprosthetic Joint Infection ◽  
Joint Infection ◽  
Next Generation ◽  
Sample Collection ◽  
Tissue Samples ◽  
Musculoskeletal Infection ◽  
Conventional Culture ◽  
Significant Difference ◽  
Generation Sequencing

Aims Use of molecular sequencing methods in periprosthetic joint infection (PJI) diagnosis and organism identification have gained popularity. Next-generation sequencing (NGS) is a potentially powerful tool that is now commercially available. The purpose of this study was to compare the diagnostic accuracy of NGS, polymerase chain reaction (PCR), conventional culture, the Musculoskeletal Infection Society (MSIS) criteria, and the recently proposed criteria by Parvizi et al in the diagnosis of PJI. Methods In this retrospective study, aspirates or tissue samples were collected in 30 revision and 86 primary arthroplasties for routine diagnostic investigation for PJI and sent to the laboratory for NGS and PCR. Concordance along with statistical differences between diagnostic studies were calculated. Results Using the MSIS criteria to diagnose PJI as the reference standard, the sensitivity and specificity of NGS were 60.9% and 89.9%, respectively, while culture resulted in sensitivity of 76.9% and specificity of 95.3%. PCR had a low sensitivity of 18.4%. There was no significant difference based on sample collection method (tissue swab or synovial fluid) (p = 0.760). There were 11 samples that were culture-positive and NGS-negative, of which eight met MSIS criteria for diagnosing infection. Conclusion In our series, NGS did not provide superior sensitivity or specificity results compared to culture. PCR has little utility as a standalone test for PJI diagnosis with a sensitivity of only 18.4%. Currently, several laboratory tests for PJI diagnosis should be obtained along with the overall clinical picture to help guide decision-making for PJI treatment. Cite this article: Bone Joint J 2021;103-B(1):26–31.

scholarly journals Next-generation sequencing-based molecular characterization of primary urinary bladder adenocarcinoma

2017 ◽  
Vol 30 (8) ◽  
pp. 1133-1143 ◽  
Author(s):  
Somak Roy ◽  
Dinesh Pradhan ◽  
Wayne L Ernst ◽  
Stephanie Mercurio ◽  
Yana Najjar ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Urinary Bladder ◽  
Molecular Characterization ◽  
Next Generation ◽  
Bladder Adenocarcinoma ◽  
Generation Sequencing

scholarly journals PENGUMPULAN DAN BATAS PEMAKAIAN SAMPEL POPOK PADA PERBENIHAN URIN

2018 ◽  
Vol 12 (2) ◽  
pp. 68
Author(s):  
Rini Riyanti ◽  
Prihatini Prihatini ◽  
M.Y Probohoesodo
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Urine Sample ◽  
Urine Culture ◽  
Practical Method ◽  
Urine Samples ◽  
Culture Result ◽  
Sample Collection ◽  
Infection Diagnosis

Urinary tract infection diagnosis is based on urine culture, taken from a midstream collection in the morning. Obtaining samplesin this manner is difficult in children less than 3 years. In children less than 3 years, urine is obtained by urine collectors. Using urinecollectors may cause discomfort, and the possibility that the urine collectors may not adhere resulting in contamination. (1) Developinga practical method for urine sample collection. (2) Comparing culture from diapers and urine collectors samples. (3) Knowing the limittime for using diapers acceptable for urine culture. Urine samples were obtained from 20 children less than 3 years, using urine collectorsand diapers used for 1 hour, 2 hours and 3 hours and then cultured. Majority of the urine culture from diapers used for 1 hour and 2hours showed the same result with the urine culture from urine collectors. Contamination was found in the urine culture result fromdiapers used for 3 hours. Urine samples from diapers used for 1 hour and 2 hours can be used as samples for urine culture. The techniqueis easy and can be done in children less than 3 years.

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