Using metagenomic methods to detect organismal contaminants in microbial materials

10.7287/peerj.preprints.2913 ◽

2017 ◽

Author(s):

Nathan D Olson ◽

Justin M Zook ◽

Jayne B Morrow ◽

Nancy J Lin

Keyword(s):

Genomic Dna ◽

Pathogen Detection ◽

A Priori ◽

High Sensitivity ◽

Chain Reaction ◽

Measurement Results ◽

Polymerase Chain ◽

Materials Used ◽

Genome Assemblies ◽

Generation Sequencing

High sensitivity methods such as next generation sequencing and polymerase chain reaction (PCR) are adversely impacted by organismal and DNA contaminants. Current methods for detecting contaminants in microbial materials (genomic DNA and cultures) are not sensitive enough and require either a known or culturable contaminant. Therefore, high sensitivity methods not requiring a priori assumptions about the contaminant are needed. We demonstrate the use of whole genome sequencing (WGS) and a metagenomic taxonomic classification algorithm for assessing the organismal purity of a microbial material. Using this proposed method we characterized the types of false positive contaminants reported and the dependence of detectable contaminant concentration on material and contaminant genome using simulated WGS data. Using the proposed method to characterize microbial material purity will help to ensure that the materials used to validate pathogen detection assays, generate genome assemblies for database submission, and benchmark sequencing methods are free of contaminants adversely impacting measurement results.

Download Full-text

Challenging a bioinformatic tool’s ability to detect microbial contaminants usingin silicowhole genome sequencing data

PeerJ ◽

10.7717/peerj.3729 ◽

2017 ◽

Vol 5 ◽

pp. e3729 ◽

Cited By ~ 6

Author(s):

Nathan D. Olson ◽

Justin M. Zook ◽

Jayne B. Morrow ◽

Nancy J. Lin

Keyword(s):

Binary Mixtures ◽

Genome Sequencing ◽

Pathogen Detection ◽

A Priori ◽

High Sensitivity ◽

False Positives ◽

Sequencing Data ◽

Materials Used ◽

The Individual ◽

Genome Assemblies

High sensitivity methods such as next generation sequencing and polymerase chain reaction (PCR) are adversely impacted by organismal and DNA contaminants. Current methods for detecting contaminants in microbial materials (genomic DNA and cultures) are not sensitive enough and require either a known or culturable contaminant. Whole genome sequencing (WGS) is a promising approach for detecting contaminants due to its sensitivity and lack of need fora prioriassumptions about the contaminant. Prior to applying WGS, we must first understand its limitations for detecting contaminants and potential for false positives. Herein we demonstrate and characterize a WGS-based approach to detect organismal contaminants using an existing metagenomic taxonomic classification algorithm. Simulated WGS datasets from ten genera as individuals and binary mixtures of eight organisms at varying ratios were analyzed to evaluate the role of contaminant concentration and taxonomy on detection. For the individual genomes the false positive contaminants reported depended on the genus, withStaphylococcus,Escherichia, andShigellahaving the highest proportion of false positives. For nearly all binary mixtures the contaminant was detected in thein-silicodatasets at the equivalent of 1 in 1,000 cells, thoughF. tularensiswas not detected in any of the simulated contaminant mixtures andY. pestiswas only detected at the equivalent of one in 10 cells. Once a WGS method for detecting contaminants is characterized, it can be applied to evaluate microbial material purity, in efforts to ensure that contaminants are characterized in microbial materials used to validate pathogen detection assays, generate genome assemblies for database submission, and benchmark sequencing methods.

Download Full-text

Evaluation of a multiplex polymerase chain reaction for early diagnosis of ventriculostomy-related infections

Journal of Neurosurgery ◽

10.3171/2014.11.jns141036 ◽

2015 ◽

Vol 123 (6) ◽

pp. 1586-1592 ◽

Cited By ~ 7

Author(s):

Claire L. Gordon ◽

Rafal Tokarz ◽

Thomas Briese ◽

W. Ian Lipkin ◽

Komal Jain ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Multiplex Polymerase Chain Reaction ◽

Pathogen Detection ◽

High Sensitivity ◽

Chain Reaction ◽

Pcr Assay ◽

Clinical Value ◽

Parallel Testing ◽

Diagnostic Approaches ◽

Polymerase Chain

OBJECT Diagnosis of ventriculostomy-related infections (VRIs) is challenging due to the lack of rapid, sensitive assays for pathogen detection. The authors report the development of a multiplex polymerase chain reaction (PCR) assay for differential diagnosis of common VRI pathogens. METHODS MassTag PCR was used to develop a multiplex assay for detection of 11 VRI pathogens. The assay was established and optimized using cloned template standards and spiked samples and was then evaluated on CSF specimens from ventricular drains. Subjects were grouped into definite VRI, possible VRI, or no VRI based on conventional microbiology, CSF evaluation, and clinical parameters. RESULTS CSF specimens were obtained from 45 subjects (median age 49 years, interquartile range 32–63 years; 51% were male). The assay detected 10–100 genome copies. It detected a pathogen in 100% (6 of 6) of definite VRI cases in which a pathogen targeted by the assay was present; these represented 67% of all definite VRIs (6 of 9). Among subjects with a possible VRI, the assay detected a pathogen in 29% (5 of 17). In subjects without overt infection the presence of a pathogen was detected in 32% of subjects (6 of 19), albeit with lower signal compared with the VRI group. CONCLUSIONS MassTag PCR enabled parallel testing of CSF specimens for 11 pathogens of VRI. The high sensitivity of PCR combined with possible device colonization, specimen contamination, and concurrent antibiotic treatments limit the clinical value of the assay, similar to other current diagnostic approaches. With further optimization, multiplex PCR may provide timely identification of multiple possible VRI pathogens and guide management, complementing classic culture approaches.

Download Full-text

Targeted Next-Generation Sequencing of Liquid Biopsy Samples from Patients with NSCLC

Diagnostics ◽

10.3390/diagnostics11020155 ◽

2021 ◽

Vol 11 (2) ◽

pp. 155

Author(s):

Hestia Mellert ◽

Jordan Reese ◽

Leisa Jackson ◽

Victoria Maxwell ◽

Chérie Tschida ◽

...

Keyword(s):

Next Generation Sequencing ◽

Liquid Biopsy ◽

High Sensitivity ◽

Next Generation ◽

Small Cell Lung ◽

Chain Reaction ◽

Targeted Next Generation Sequencing ◽

Polymerase Chain ◽

Test Process ◽

Generation Sequencing

Liquid biopsy tests have become an integral part of the molecular diagnosis of patients with non-small cell lung cancer (NSCLC). We describe a new test panel that uses very low input (20 ng) of cell-free nucleic acids extracted from human plasma, which is designed to yield results in less than 72 h. In this study, we performed novel amplicon-based targeted next-generation sequencing with a semiconductor-based system, the Ion GeneStudio S5 Prime. The analytic performance of the assay was evaluated using contrived and retrospectively collected clinical specimens. The cumulative percent coefficient of variation for the new test process was very precise at 8.4% for inter-day, 4.0% for inter-operator and 3.4% for inter-instrument. We also observed significant agreement (95.7–100%) with an orthogonal, high-sensitivity droplet digital™ Polymerase Chain Reaction (ddPCR) test. This method offers a valuable supplement to assessing targeted mutations from blood while conserving specimens and maintaining sensitivity, with rapid turn-around times to actionable results.

Download Full-text

Analysis of Bacteria Community in the sediment from Bangka Island, North Sulawesi

JURNAL ILMIAH PLATAX ◽

10.35800/jip.8.2.2020.29887 ◽

2020 ◽

Vol 8 (2) ◽

pp. 196

Author(s):

Meivyarni Wangka ◽

Stenly Wullur ◽

Esther Angkouw ◽

Jane Mamuaja ◽

Reiny Tumbol ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Next Generation Sequencing ◽

Nucleic Acid ◽

Genomic Dna ◽

Freezing And Thawing ◽

Chain Reaction ◽

Bacterial Dna ◽

North Sulawesi ◽

Polymerase Chain ◽

Generation Sequencing

Marine sediments are nutrient-rich and is a suitable habitat of bacteria. This research is a preliminary study of molecular analysis to identify the bacteria in the sediments from the littoral area that covered by mangroves in Bangka Island, North Sulawesi. The purposes of this study are to obtain the uncultivated bacterial DNA genome which is used to identify the bacteria and bacterial community in the sediments. Isolation of DNA genome from uncultured bacterial was carried out by following the genomic DNA extraction procedure using the DNeasy® PowerSoil Extraction Kit. Before isolating the bacterial DNA, sample were went through freezing and thawing processes. The DNA isolation result was subsequently tested using electrophoresis and UV-Vis spectrophotometers. Subsequently the genomic DNA was amplified using Polymerase Chain Reaction and the bacteria were identified using Next Generation Sequencing (NGS) analysis. The results of this study showed that the DNA of uncultured bacteria from sediment have the purity of 1.05 and the DNA amplification band was detected at 1300-1600bp. The bacteria in Bangka Island, North Sulawesi sediments were consisted of Gemmatimonadetes, Acidobacteria, Chioroflexi, Firmicutes, Bacteroidetes, Actinobacteria, Cyanobacteria and Proteobacteria respectively. Phylum Proteobacteria was found has the highest relative abundance in the sediment.Keywords : Bacteria, Deoxyribo Nucleic Acid, Sediment, Uncultured. ABSTRAKSedimen laut merupakan suatu habitat yang kaya akan nutrient dan merupakan habitat dari bakteri. Penelitian ini merupakan tahapan awal dalam rangkaian analisis molekuler bakteri yang hidup di sedimen dari daerah litoral yang ditumbuhi oleh mangrove pada Pulau Bangka Sulawesi Utara. Tujuan penelitian ini adalah untuk mendapatkan DNA genom bakteri tanpa kultivasi yang digunakan dalam analisis jenis dan komunitas bakteri pada sedimen. Isolasi DNA genom bakteri tanpa kultivasi dilakukan dengan mengikuti prosedur Kit ekstraksi DNA DNeasy® PowerSoil. Sebelum tahap isolasi DNA bakteri, sampel diperlakukan proses freezing and thawing. Hasil isolasi DNA diuji menggunakan elektroforesis dan spektrofotometer UV-Vis. DNA genom diamplifikasi menggunakan Polymerase Chain Reaction dan ditentukan jenis bakteri dengan menggunakan Next Generation Sequencing analysis. Hasil penelitian menujukkan bahwa DNA bakteri tanpa kultivasi memiliki kemurnian 1,05. DNA amplifikasi terdeteksi pada posisi 1300-1600bp. Dan jenis bakteri yang hidup pada sedimen di Pulau Bangka Sulawesi Utara, terdiri dari filum Gemmatimonadetes, Acidobacteria, Chioroflexi, Firmicutes, Bacteroidetes, Actinobacteria, Cyanobacteria dan Proteobacteria. Kelimpahan tertinggi bakteri yang hidup pada sedimen tersebut adalah filum Proteobacteria.Kata kunci : Bakteri, Deoxyribo Nucleic Acid, Sedimen, Tanpa kultivasi.

Download Full-text

Use of Multiple Displacement Amplification as Pre-polymerase Chain Reaction (Pre-PCR) to amplify genomic DNA of siphonapterids preserved for long periods in scientific collections

Parasites & Vectors ◽

10.1186/1756-3305-3-86 ◽

2010 ◽

Vol 3 (1) ◽

pp. 86 ◽

Cited By ~ 5

Author(s):

Daniel M Avelar ◽

Pedro M Linardi

Keyword(s):

Polymerase Chain Reaction ◽

Genomic Dna ◽

Multiple Displacement Amplification ◽

Chain Reaction ◽

Polymerase Chain ◽

Scientific Collections

Download Full-text

Analysis of glutathione S-transferase genes polymorphisms and the risk of schizophrenia in a sample of Iranian population

Neuron Glia Biology ◽

10.1017/s1740925x12000130 ◽

2011 ◽

Vol 7 (2-4) ◽

pp. 199-203 ◽

Cited By ~ 7

Author(s):

Farah Lotfi Kashani ◽

Dor Mohammad Kordi-Tamandani ◽

Roya Sahranavard ◽

Mohammad Hashemi ◽

Farzaneh Kordi-Tamandani ◽

...

Keyword(s):

Genomic Dna ◽

Case Control Study ◽

Gene Interaction ◽

Case Control ◽

Glutathione S Transferase ◽

Chain Reaction ◽

Healthy Control ◽

Glutathione S Transferases ◽

Polymerase Chain ◽

Control Study

Glutathione S-transferases (GSTs) are major intracellular antioxidants, which, impaired in their function, are involved in the progress of schizophrenia (SCZ). The aim of this case-control study was to investigate the association between the polymorphism of glutathione S-transferases M1 (GSTM1), T1 (GSTT1), the glutathione S-transferase P1 gene (GSTP1) and SCZ. We isolated genomic DNA from peripheral blood of 93 individuals with SCZ and 99 healthy control subjects' genotypes analyzing them for GSTM1, GSTT1 and GSTP1 using polymerase chain reaction. The analysis of the gene–gene interaction between GSTs indicated that the magnitude of the association was greater for the combined AG/GSTT1 & GSTM1 genotypes (OR = 2.51; 95% CI: 1.13–5.63, P = 0.02). The AG and combined AG + GG genotypes of GSTP1 increased the risk of SCZ (OR = 1.83; 95% CI: 0.94–3.75 and OR = 1.71; 95% CI: 0.92–3.19, respectively). The genotypes of GSTT/NULL, NULL/GSTM and NULL/NULL increased the risk of SCZ (OR = 2.05; 95% CI: 0.9–4.74; OR = 2.0; 95% CI: 1.68–2.31; and OR = 1.8; 95% CI: 0.57–2.46, respectively). The present study supports previous data that suggest that impairment in the function of GSTs genes may increase the risk of SCZ.

Download Full-text

Use of the polymerase chain reaction for detection of Fusarium graminearum in bulgur wheat

Food Science and Technology ◽

10.1590/s0101-20612012005000027 ◽

2012 ◽

Vol 32 (1) ◽

pp. 201-208 ◽

Cited By ~ 1

Author(s):

Carla Bertechini Faria ◽

Giovana Caputo Almeida-Ferreira ◽

Karina Bertechine Gagliardi ◽

Tatiane Cristina Albuquerque Alves ◽

Dauri José Tessmann ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Fusarium Graminearum ◽

Genomic Dna ◽

Solid Medium ◽

Food Contamination ◽

Chain Reaction ◽

Specific Pcr ◽

Culture Characteristics ◽

Mycotoxigenic Fungi ◽

Polymerase Chain

The detection of mycotoxigenic fungi in foodstuff is important because their presence may indicate the possible associated mycotoxin contamination. Fusarium graminearum is a wheat pathogen and a producer of micotoxins. The polymerase chain reaction (PCR) has been employed for the specific identification of F. graminearum. However, this methodology has not been commonly used for detection of F. graminearum in food. Thus, the objective of the present study was to develop a molecular methodology to detect F. graminearum in commercial samples of bulgur wheat. Two methods were tested. In the first method, a sample of this cereal was contaminated with F. graminearum mycelia. The genomic DNA was extracted from this mixture and used in a F. graminearum specific PCR reaction. The F. graminearum species was detected only in samples that were heavily contaminated. In the second method, samples of bulgur wheat were inoculated on a solid medium, and isolates having F. graminearum culture characteristics were obtained. The DNA extracted from these isolates was tested in F. graminearum specific PCR reactions. An isolate obtained had its trichothecene genotype identified by PCR. The established methodology could be used in surveys of food contamination with F. graminearum.

Download Full-text