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Scientific Publications on NGS Applications in Microbiology and Infectious Disease Diagnostics

This page introduces a selection of scientific publications on next generation sequencing (NGS) applications in microbiology and infectious diseases including research studies and diagnostics. Publications related to animal genetics (health) and food safety are introduced in the corresponding category. 


General Infection Diagnostic Publications

A method for amplicon deep sequencing of drug resistance genes in Plasmodium falciparum clinical isolates from India

23. March 2016. The Journal of Clinical Microbiology published the study from Roa et al. "A method for amplicon deep sequencing of drug resistance genes in Plasmodium falciparum clinical isolates from India."
This publication describes the development of a novel NGS targeted gene panels for six Plasmodium falciparum genes related to antimalarial therapy resistance including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine.
The targeted genes are Pfk13 (resistance to artemisinin), Pfcrt (chloroquine resistance transporter), Pfdhfr (dihydrofolate reductase) and Pfdhps (dihydropteroate synthetase) both associated with resistance against antifolates sulfadoxine and pyrimethamine, Pfmdr1 (multidrug resistance protein-1) and Pfmrp1 (plasma membrane ABC transporter) associated with resistance to multiple antimalarial drugs.
The protocol was evaluated on 12 P. falciparum reference strains and tested with multiplexed patient samples. Previously described known SNPs associated with drug resistance were reliable detected in these reference strains by applying the new NGS panel. Furthermore, sequencing of the complete genes enables the detection of unknown SNPs related to drug resistance.
In summary, this new method could be implemented in national malaria control programs as multiplexing of samples enable reasonable cost per sample and the utilization of Ion Torrent PGM technology enables data analysis without advanced bioinformatics trainings.
The publication is free available on Journal of Clinical Microbiology

A Next-Generation Sequencing Data Analysis Pipeline for Detecting Unknown Pathogens from Mixed Clinical Samples and Revealing Their Genetic Diversity

17. March 2016, PlosOne published the study from Gong et al." A Next-Generation Sequencing Data Analysis Pipeline for Detecting Unknown Pathogens from Mixed Clinical Samples and Revealing Their Genetic Diversity."
In this study 42 CPE positive clinical samples were investigated in which pathogens could not be detected by routine diagnostics. Next generation sequencing was applied and a data analysis pipeline was proposed to detect unknown pathogens in mixed samples. In all samples viral pathogens could be detected by NGS and 40 samples could be confirmed by PCR or ELISA afterwards. The two missing strains were predicted as human parechovirus (HPeV) by NGS data analysis.
Iterative mapping procedure was implemented in order to construct viral genomes in cases without genomic reference.
Phylogenetic and recombination analysis of the HPeV strains was performed. The data were used to predict the genetic evolution of HPeV. Furthermore, data analysis by Simplot and Bootscan demonstrates the occurrence of recombination in multiple HPeV strains.
In summary, the proposed NGS data analysis pipeline could detect unknown viruses from mixed samples by NGS that were not detected with routine diagnostics. It has to be mentioned that the clinical samples were cultured prior NGS analysis.
The publication is free available on PubMed

DNAseq Workflow in a Diagnostic Context and an Example of a User Friendly Implementation

This publication introduces GenesearchNGS, a commercial software suite for NGS data analysis. The software was developed specifically for data analysis in a diagnostic context, and so the focus of the publication lies in NGS diagnostics applications, especially for SNP detection and analysis. The authors describe the steps required for NGS data analysis in diagnostics, along with the GenesearchNGS workflow and analysis tools. GenesearchNGS enables distributed sequence alignment by connecting different computers within an existing network or by using the Amazon cloud server.
In summary, GenesearchNGS addresses limitations experienced by smaller laboratories, such as user friendly data analysis and the lack of bioinformatics knowledge and computing capacity.
Wolf et al 2015

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The role of host genetic factors in respiratory tract infectious diseases: systematic review, meta-analyses and field synopsis

This is the first extensive meta analysis of five common respiratory tract infectious diseases. In this publication a meta data analysis on studies of tuberculosis, influenza, respiratory syncytial virus, SARS-Coronavirus and pneumonia was conducted in order to identify host genetic factors.
Numerous studies on infectious agents and host interaction were conducted in order to find out more about host genetic factors, potential issues of this studies are discussed.
Two genetic host factors (IL4 and TLR2) were discovered. Additionally, CCL2 could play a role during tuberculosis infection. Limiting factors of this study and the primary studies are extensive discussed.
Patarčić et al. 2015

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Publications of NGS applications in Bacteriology

A complete bacterial genome assembled de novo using only nanopore sequencing data

In this brief communication, the authors report complete E. coli genome assembling using Nanopore sequencing technology. The challenges of long read data analysis are discussed, and applicable software tools are introduced. The author verifies the possibility of using Nanopore technology for sequencing and assembling complete bacterial genomes without the use of addition short read sequencing methods. The application of signal-level algorithms was shown to further improve assembling accuracy.
Loman et al. 2015


An evaluation of alternative methods for constructing phylogenies from whole genome sequence data: a case study with Salmonella.

In this study, three key factors for the construction of phylogenetic relationships were investigated in whole genome sequencing data of 194 Salmonella samples. These factors are the NGS platform, the algorithm used for SNP constructing, and phylogenetic inference.
A comparison of NGS platforms confirmed the previously documented differences in accuracy when using MySeq, Roche 454, and Ion Torrent to identify the lowest single base error rate. The reference-free variant identification method returned more false positive SNPs and produced more incongruent trees compared with the reference-based methods. The results from the three phylogenetic inference methods showed that a high degree of accuracy correlates with a trade-off in speed.
Pettengill et al 2014

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Chronic Psychological Stress Disrupted the Composition of the Murine Colonic Microbiota and Accelerated a Murine Model of Inflammatory Bowel Disease

7. March 2016, PlosOne published the study of Watanabe et al. "Chronic Psychological Stress Disrupted the Composition of the Murine Colonic Microbiota and Accelerated a Murine Model of Inflammatory Bowel Disease".

In this study the relationship between water-avoidance stress (over 12 weeks) and the murine colonic microbiota was investigated in T cell receptor alpha chain gene knockout mouse by 16S rRNA genes sequencing.
The study showed that knockout of the Tcra gene disrupts the microbiota balancing systems, leading to an unstable microbiota.
These findings suggest that imbalances in the colonic microbiota (loss of phylogenetic diversity) combined with propagation of specific endogenous pathogens are important factors for the development of stress-induced aggravation of colitis.
Furthermore, sensitive mice with decreases colonic microbiota phylogenetic diversity showed increased luminal IgA levels. This is in accordance with previously shown results that IgA regulate the diversity and composition of the colonic microbiota.
In summary, the results showed that a high phylogenetic diversity in the colonic microbiota can be an important factor for preventing increases in disease activity induced by chronic psychological stress in inflammatory bowel disease.

The publication is free available on PlosOne

Comparison of the Oral Microbiomes of Canines and Their Owners Using Next-Generation Sequencing

In this study, 16S rDNA pyrosequencing was used to compare the oral microbiomes of 10 dogs and their owners, in order to identify zoonotic pathogens. In summary, bacterial transmission from dogs to humans was found to be limited.
Oh et al. 2015

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Metagenomics: A New Way to Illustrate the Crosstalk between Infectious Diseases and Host Microbiome

This review focuses on the current knowledge of the alteration of complex microbial communities upon the infection of different pathogens, such as human immunodeficiency virus, hepatitis B virus, influenza virus, and Mycobacterium tuberculosis, at different body sites.
This review starts with an introduction to the human microbiome and different NGS approaches for metagenome analysis.
The alteration of the microbiome of HIV patient (with different treatments or without treatment) was investigated and is described in more detail for different body sides such as the Gastrointestinal Tract, the rectal side, the genital tract, or blood, semen and brain as well as in Oral Cavity and Airway.
Furthermore, the alteration of the human microbiome after infection with tuberculosis is discussed in more detail.
As a conclusion, the authors stat that the metagenomics approach will be beneficial over the widely used 16sRNA based targeted approach, as metagenomics also detects viruses and other microorganism whereas the 16sRNA approach only detects bacteria. Furthermore, the metagenomics approach will provide additional information about functionality and can provide information about growth dynamics.
Zhang et al. 2015


Sequencing of Bacterial Genomes: Principles and Insights into Pathogenesis and Development of Antibiotics

This review presents a brief overview of bacterial pathogenesis followed by an explanation of interactions between bacteria and antibiotics and a brief introduction to the bacterial genome. Bacterial genome sequencing is discussed, using the example Streptococcus Pneumoniae TIGR4. Furthermore, the application of NGS technology to pathogenic genome analysis, such as the identification and comparison of pathogenic islands, is described, along with genome sequencing applications for the development of novel antibiotics.
In summary, the review demonstrates how advances in sequencing have greatly improved the understanding of bacterial pathogenic mechanisms. New antibiotics based on progress in bacterial genomic sequencing have hardly reached the market, however.
E. S. Donkor 2013

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Investigation of microbial composition in marine sediments by next generation sequencing

Choi et al. published in the Journal of Microbiology and Biotechnology the study "Microbial community composition in the marine sediments of Jeju Island: next-generation sequencing surveys".
This study was performed in order to investigate the structure of the archaeal and bacterial communities in the surface and subsurface sediments of Jeju Island.
For bacteria classification the 16S rRNA gene was amplified by PCR prior next generation pyrosequencing.
The results showed that the archaeal and bacterial diversity of the subsurface
sediments were slightly higher than those for the surface sediments. Twelve different archaeal groups could be detected by NGS. Twenty six different phyla including an unclassified bacterial group were found in the surface and subsurface sediments.
Although limited genetic information was available reads that were affiliated with functional genera of bacteria could be identified.
The publication is free available at PMC.

Publications of NGS applications in Virology

Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

This review begins with an introduction to NGS technologies and follows with an overview of NGS applications in virus diagnostics. The NGS applications discussed are the detection of unknown viruses and discovery of new viruses, detection of tumour viruses, characterisation of the human virome, investigation of viral genome variability and quasispecies analysis, monitoring of antiviral drug resistance, viral epidemiology and evolution, and quality control of vaccines.
Furthermore, the author addresses the challenges arising during NGS data analysis.
In summary, this review provides a solid overview of NGS applications in diagnostic virology, despite its 2011 publication date.
Barzon et al. 2011

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Application of next-generation sequencing technologies in virology

The author gives a brief introduction to the major NGS technologies, including Illumina, Ion Torrent, Helicos, Roche454, SOLiD™, and PacBio. Differences between the technologies and their applications are described, followed by an overview of sample preparation. NGS application in virology, including targeted amplification of viruses, viral metagenomics, virus candidate pathogen detection, environmental viral metagenomics and transcriptomics are also reviewed.
Radford et al 2012

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Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification

This publication examines the sequence independent amplification of ultra-low virus samples, such as West Nile Fever, HIV, and respiratory syncytial virus, using Illumina technology. This publication focuses on NuGEN’s Ovation RNA-Seq system for sequence-independent amplification. Both versions (1 and 2) of the system were tested and compared. In this study successful RNA virus sequencing from low concentration samples with 100 viral genomes was demonstrated. Furthermore, the complete sequence coverage of the CDS from clinical samples was shown. For assembling of genomes, the VICUNA assembler was described for the first time.
Malboeuf et al 2012

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Detection of Arboviruses and Other Micro-Organisms in Experimentally Infected Mosquitoes Using Massively Parallel Sequencing

This study sought to prove the concept that NGS technology can be applied to the investigation of mosquito infections with different viruses such as DENV, VNF or CHIKV. The results demonstrated that, not only the virus was detected by NGS, but that also information on the host genome and associated microorganism could be obtained. The associated endosymbiotic Wolbachia bacterium could be classified at the species level using this process.
Hall-Mendelin et al 2013

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Development of a virus detection and discovery pipeline using next generation sequencing

The development of a new pipeline for virus identification from NGS data, with a focus on plant samples, is described in this paper. This pipeline includes the novel virus detection software tool VirFind, and was shown to be capable of detecting all known plant viruses, as well as some unknown plant viruses.
Furthermore, this pipeline can also be used to detect virus from any other host.
Ho and Tzanetakis 2014


Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

The study was conducted in order to develop a new simplified tool for whole genome sequencing using next generation sequencing of influenza A viruses. The developed method is based on the already described multisegment reverse transcription-PCR (M-RTPCR) approach. In this approach eight genomic RNA segments of the influenza A virus are amplified in parallel in one reaction. In this study the sensitivity of the M-RTPCR method for its application in subsequent next generation sequencing was evaluation on multiple and mixed influenza samples. The results showed that mixed influenza subtypes could reliable be sequenced also in different dilutions. This method enabled complete sequencing of low titer influenza genomes and partially sequencing sufficient for subtyping of very low influenza A virus. The method was also tested on clinical samples and enables the sequencing of clinical samples completely or partially depending on the virus titer.
In summary, the investigated approach enables the sequencing of influenza A virus in a single reaction and allows discrimination of different influenza A subtypes in one sample if the virus titer is adequate.

Zou et al. 2016

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HIV-1 neutralizing antibody response and viral genetic diversity characterized with next generation sequencing

In this study, HIV neutralising antibody titre breadth was compared with HIV virus diversity. A number samples from HIV infected patients were investigated at different time points in order to determine whether HIV diversity correlated with the neutralising antibody titre breadth. Antibody response against env, gag and pol viral proteins were also compared.
Carter et al 2015


Implementation of next-generation sequencing for hepatitis B resistance and genotyping in a clinical microbiology laboratory

This publication introduces the currently used methods for HBV diagnosis and genotyping INNO-LiPA (LiPA), Sanger sequencing, restriction fragment polymorphism (RFLP), and PCR. The advantages and disadvantages compared to next generation sequencing are discussed.
Next generation sequencing can overcame limitations of Sanger sequencing (lack of sensitivity) and LiPA (not able to detect new mutations).
In summary, NGS enables high sensitivity in HBV genotyping and resistance testing. The high costs and limited guidelines for clinical testing are still obstacles for the implementation of NGS in HBV resistance and genotyping diagnostics.
The major benefit of NGS in HBV diagnostics is the detection of novel and low level variants.

Lowe et al. 2015

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Next-generation sequencing technologies in diagnostic virology

This publication provides suggestions for virologists planning to implement NGS technology in their research. The author discusses some standard considerations for establishing a NGS lab, including the proposed field of NGS application, the technology selected, cost, accuracy, throughput, read length, speed, upgrade capacity, and automation. Different pitfalls of NGS applications in diagnostics are listed, and major applications for NGS in diagnostics are discussed, such as identification of novel viruses, metagenome analysis of human virome, full-length viral genome sequencing, viral population analysis, and virus host interaction analysis. Finally, the challenges in data analysis and bioinformatics are briefly discussed.
Overall, this review provides a concise and practical introduction to NGS applications and considerations in diagnostic virology.
Barzon et al. 2013


Phylogenetic evidence for intratypic recombinant events in a novel human adenovirus C that causes severe acute respiratory infection in children

10. March 2016, Nature published the study fo Wang et al. "Phylogenetic evidence for intratypic recombinant events in a novel human adenovirus C that causes severe acute respiratory infection in children"
This publication reports on a recombinant HAdV strain (CBJ113) isolated from a children with severe acute respiratory infection (SARI). By applying NGS a novel adenovirus subtype HAdV-C was analyzed and the prevalence in China was estimated to 4.7% of all HAdV infections.
The phylogenetic analysis of this novel strain showed a common origin with different known HAdV strains and formed a new subclade with other HAdV-C subtypes.
The detailed analysis proved that certain genome regions showed high sequence similarities with certain HAdV strains. In order to investigate the occurrence of recombination, similarity plot and BootScan analyses were performed with the SimPlot software. The analysis of this new HAdV-C strain indicated a mosaic pattern arose from recombination events with different HAdV strain. These results were confirmed by SNP pattern analysis.
In summary, a new HAdV strain was isolated in China that displays a mosaic pattern of genetic information from different HAdV strains caused by recombination. The epidemiologic surveillance study indicates that this strain is already prevalent in China for a long time.
The publication is free available at Nature

Rapid spread of influenza A(H1N1)pdm09 viruses with a new set of specific mutations in the internal genes in the beginning of 2015/2016 epidemic season in Moscow and Saint-Petersburg (Russian Federation)

19. March 2016, Wiley Online published the study of Komissarov et al. "Rapid spread of influenza A(H1N1)pdm09 viruses with a new set of specific mutations in the internal genes in the beginning of 2015/2016 epidemic season in Moscow and Saint-Petersburg (Russian Federation)."
This publication reports on influenza A(H1N1) pdm09 cases from early 2016 in Russia. This influenza activity differed significant from previous seasons in higher incidence of acute respiratory infection (ARI), influenza-like illness (ILI), and number of lethal cases.
The antigenic analysis of this Russian influenza A(H1N1) pdm09 strain showed high similarity with the vaccine strain.
The whole genome sequencing analysis of 77 different isolated strains from this season displayed several substitutions in the neuraminidase gene, furthermore a number of substitutions in internal genes were detected.
Phylogenetic analysis suggested that the most relevant influenza strain bearing similar substitutions was isolated in New York in August 2015.
The observed rapid spread of the new influenza A(H1N1)pdm09 in Russia was explained with improved transmissibility or virulence. This assumption need to be confirmed by experimental data.
The publication is free available on Wiley Online Library

Spouse-to-Spouse Transmission and Evolution of Hypervariable Region 1 and 5’ Untranslated Region of Hepatitis C Virus Analyzed by Next-Generation Sequencing

26. February 2016, PlosOne published the study from Cortes et al. " Spouse-to-Spouse Transmission and Evolution of Hypervariable Region 1 and 5’ Untranslated Region of Hepatitis C Virus Analyzed by Next-Generation Sequencing"

This study was conducted in order to investigate the transmission and selection of HCV variants from a chronically infected female donor to a male (spouse) recipient applying next generation sequencing technology. The study focus on two genetic regions, the hyper variable region (HVR1) and the 5´untranslated region (5´UTR). The donor was chronically infected for 10 years and the recipient developed an acute infection evolving to chronic hepatitis. Serum samples were investigation during the acute infection phase and at different time points afterwards. The results were compared with 10 randomly selected samples of chronic infected patients with the same HCV subtype.
Haplotype prediction was performed by applying Shorah software.
Results proved a higher sequence similarity between the donor and the recipient compared to the HCV subtype sequenced in the randomly selected patients. The detection of minor donor variants could only be proofed by applying next generation sequencing based analysis. Molecular evolution of HVR1 was different between the chronically infected donor and the acute infected recipient. The 5´UTR region was more stable probably due to less selective pressure.
In conclusion intra-familial HCV transmission may be established by minor variants whose investigation requires sensitive analysis tools such as NGS.
The publication is free available at PlosOne

Zahedan rhabdovirus, a novel virus detected in ticks from Iran

This publications introduced the findings of a novel rhabdovirus from Hyalomma anatolicum anatolicum ticks found in Iran. Almost the complete genome of the newly named Zahedan rhabdovirus (ZARV) was sequenced using 454 technology after cultivation of the virus in vero cell culture.
The data were analysed and phylogenetic studies with related viruses were conducted. The new ZARV is closely related to MOUV and LITRV. Additionally, a conventional RT-PCR assay for detection of the ZARV was developed and evaluated. Results of this study lead to the expectation that the virus might infect mammalians. Information about the mammalian host are still missing.
Dilcher et al. 2015

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Publications of NGS applications in phages

Safety analysis of a Russian phage cocktail: From MetaGenomic analysis to oral application in healthy human subjects

According to the World Health Organization, a global antibiotic crisis exists due to increasing numbers of resistant bacteria and a dearth of new antibiotics under development by the pharmaceutical industry. Thus, there is an urgent need for alternative strategies to antibiotics. Bacteriophage therapy is a promising treatment and very common in some regions, such as the Russian Federation. In this study, the phage composition of a Russian commercially available phage cocktail, ColiProteus, was investigated by NGS and 17different groups of phages were detected. In order to evaluated the safety of the phage cocktail, human volunteers of different ages were treated. No significant side effects were observed, but reliable data on the efficiency of the phage cocktail during bacterial infection are not yet available.
McCallin et al 2013

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